No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells
Abstract
A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.
... Bcl-2 overexpression has been associated with accelerated G1 arrest and delayed G1/S transition. Owing to growth factor deprivation, the cells presenting high BCL2 levels were smaller than the controls 40 . The lack of c-myc expression was also correlated with a reduction in nuclear size, which is considered an indicator of cell cycle arrest in G0 by BCL2-positive cells. ...
... The lack of c-myc expression was also correlated with a reduction in nuclear size, which is considered an indicator of cell cycle arrest in G0 by BCL2-positive cells. This allows maintaining cell viability in abnormal conditions 40 . This is in line with BCL2 antiapoptotic function 41 . ...
Carnosol, a rosemary polyphenol, displays anticancer properties and is suggested as a safer alternative to conventional surgery, radiotherapy, and chemotherapy. Given that its effects on gingiva carcinoma have not yet been investigated, the aim of this study was to explore its anti-tumor selectivity and to unravel its underlying mechanisms of action. Hence, oral tongue and gingiva carcinoma cell lines exposed to carnosol were analyzed to estimate cytotoxicity, cell viability, cell proliferation, and colony formation potential as compared with those of normal cells. Key cell cycle and apoptotic markers were also measured. Finally, cell migration, oxidative stress, and crucial cell signaling pathways were assessed. Selective anti-gingiva carcinoma activity was disclosed. Overall, carnosol mediated colony formation and proliferation suppression in addition to cytotoxicity induction. Cell cycle arrest was highlighted by the disruption of the c-myc oncogene/p53 tumor suppressor balance. Carnosol also increased apoptosis, oxidative stress, and antioxidant activity. On a larger scale, the alteration of cell cycle and apoptotic profiles was also demonstrated by QPCR array. This was most likely achieved by controlling the STAT5, ERK1/2, p38, and NF-ĸB signaling pathways. Lastly, carnosol reduced inflammation and invasion ability by modulating IL-6 and MMP9/TIMP-1 axes. This study establishes a robust foundation, urging extensive inquiry both in vivo and in clinical settings, to substantiate the efficacy of carnosol in managing gingiva carcinoma.
... The therapeutic targeting of Bcl-2 in leukemia emerged following the elucidation of its crucial role in evading apoptosis. Bcl-2 was initially associated with a proliferative function upon its discovery in follicular lymphoma translocation (14:18), leading to its constitutive activation by enhancers of the immunoglobulin heavy chain locus [26]. It was subsequently delineated that Bcl-2 belongs to the broader BCL family of proteins, orchestrating a delicate balance between apoptosis and cell survival. ...
... Venetoclax has become the only selective BH3 mimetic currently approved by the United States Food and Drug Administration (FDA) for the treatment of CLL/SLL in adult patients and, in combination with azacitidine, decitabine, or low-dose cytarabine, for newly diagnosed AML in adults 75 years or older, or who have comorbidities precluding intensive induction chemotherapy [39,40]. tion upon its discovery in follicular lymphoma translocation (14:18), leading to its consti-tutive activation by enhancers of the immunoglobulin heavy chain locus [26]. It was subsequently delineated that Bcl-2 belongs to the broader BCL family of proteins, orchestrating a delicate balance between apoptosis and cell survival. ...
Venetoclax is a Bcl-2 homology domain 3 (BH3) mimetic currently approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) that has proven to be highly effective in reinstating apoptosis in leukemic cells through the highly selective inhibition of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). Clinically, venetoclax has provided lasting remissions through the inhibition of CLL and AML blasts. However, this activity has often come at the cost of grade III/IV neutropenia due to hematopoietic cells’ dependence on Bcl-2 for survival. As life-threatening infections are an important complication in these patients, an effective management of neutropenia is indispensable to maximize patient outcomes. While there is general consensus over dose reduction and scheduling modifications to minimize the risk of neutropenia, the impact of these modifications on survival is uncertain. Moreover, guidelines do not yet adequately account for patient-specific and disease-specific risk factors that may predict toxicity, or the role combination treatment plays in exacerbating neutropenia. The objective of this review is to discuss the venetoclax-induced mechanism of hematological toxicity, the potential predictive risk factors that affect patient vulnerability to neutropenia, and the current consensus on practices for management of neutropenia.
... Избыточное количе ство фактора у взрослых животных вызывало ингиби рование пролиферации дендритных клеток, атрофи ческие изменения в тимусе с подавлением развития Тлимфоцитов [5]. Добавление VEGFA в колонию гемопоэтических стволовых клеток приводило к уве личению экспрессии антиапоптотических генов (на пример, bcl-2) in vitro [2,18]. Фактор в повышенной концентрации может оказывать стимулирующее и анти апоптотическое действие, способствуя процессам лей кемогенеза. ...
... По результатам большого количества научных ис следований отмечается увеличение экспрессии гена VEGF в бластных клетках пациентов с острым лимфо бластным лейкозом и ОМЛ [4,18,[25][26][27][28]. Выявлено увеличение концентрации белка VEGFA в лизатах бластных клеток крови и костного мозга пациентов с ОМЛ [29]. ...
Background. Vascular endothelial growth factor A (VEGFA) is one of the most important factors for regulation of hematopoietic stem cells differentiation. It is involved in leukemogenesis and central nervous system (CNS) damage in acute leukemia. According to the literature, the VEGFA production by blast cells is increased, but the values of serum concentration and the associations with CNS involvement are contradictory.
Aim. evaluate the VEGFA, VEGFR1, VEGFR2 concentration in serum and cerebrospinal fluid of patient with different types of acute leukemia in disease onset and during treatment.
Materials and methods. The concentration of VEGFA in serum and cerebrospinal fluid was studied in 74 primary patients with acute leukemia. The comparison group consisted of 67 healthy donors. VEGFR1, VEGFR2 were studied in serum and cerebrospinal fluid in 34 patients at the onset of the disease. The comparison group consisted of 10 healthy donors. For the analysis, an enzyme immunoassay was used on a semi-automatic Personal Lab analyzer (Adaltis) and Affymetrix eBioscience human VEGF-A Platinum ELISA reagents.
Results. Serum VEGFA concentration was statistically significantly lower in acute leukemia patients than that of donors (median 149.78 and 432.19 pg/ml respectively; p <0.0001). Factor deficiency was significantly more pronounced in patients with blastemia ( p <0.015). During antitumor therapy, there was a tendency to increase the amount of the factor in the blood serum. Serum concentration of soluble VEGFR2 was also lower in patients than that of donors (6949.9 and 8795.9 pg/ml respectively; p = 0.0026). For concentration of VEGFR1 such deviations were not found. The concentrations of VEGFR1 and VEGFR2 in serum were higher than in cerebrospinal fluid ( p <0.0001), while VEGFR1 showed a positive correlation between serum and cerebrospinal fluid concentrations. the concentration of VEGFR1 in the cerebrospinal fluid was significantly lower in patients with B-lymphoblastic leukemia/lymphoma compared to other types of leukemia.
Conclusion. the concentration of VEGFA in serum decreases in patients with blastemia, this may indicate a lack of secretion and excessive consumption of the factor by blast cells with a decrease in the proportion of leukocytes that normally secrete the factor. In the cerebrospinal fluid, the concentrations of VEGFR1 and VEGFR2 are lower than in serum, with the lowest values being found in patients with B-lymphoblastic leukemia/lymphoma, but no relationship with the development of CNS involvement was found.
... BCL2 protein is known to exert anti-apoptotic effects and its expression is usually upregulated in tumor cells [16]. Quantitative RT-PCR revealed that PRF with MG63 led to a significant down-regulation of the relative gene expression of BCL2 as compared to the control ( Figure 4D). ...
... Statistical significance was assessed when * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, ns = not significant. BCL2 protein is known to exert anti-apoptotic effects and its expression is usually up-regulated in tumor cells [16]. Quantitative RT-PCR revealed that PRF with MG63 led to a significant down-regulation of the relative gene expression of BCL2 as compared to the control ( Figure 4D). ...
The understanding that tumor cells might evade immunity through various mutations and the potential of an augmented immune system to eliminate abnormal cells led to the idea of utilizing platelet-rich fibrin (PRF), a blood concentrate containing the body’s immune elements as an adjunctive therapy for localized tumors. This study is the first that evaluated the effect of PRF generated with different relative centrifugal forces (RCFs) on osteoblastic and fibroblastic tumor cell lines MG63 and HT1080 with regard to cell viability, cytokine and growth factor release, and the gene expression of factors related to the cell cycle and apoptosis. Our findings could demonstrate decreased cell proliferation of MG63 and HT1080 when treated indirectly with PRF compared to cell cultures without PRF. This effect was more distinct when the cells were treated with low-RCF PRF, where higher concentrations of growth factors and cytokines with reduced RCFs can be found. Similar patterns were observed when assessing the regulation of gene expression related to the cell cycle and apoptosis in both MG63 and HT1080 cells treated with PRF. Despite variations, there was a consistent trend of an up-regulation of tumor-suppressive genes and a down-regulation of anti-apoptotic genes in both cell types following treatment with high- and, particularly, low-RCF PRF formulations.
... In the era of precision medicine, there is a search for markers that could provide more precise information about the risk of recur-rence and progression of NIMBC. Several promising molecular markers were studied, such as BCL2, TP53, FOXA1, and GATA3 often with contradictory results (11)(12)(13)(14)(15). ...
... The balance between Bcl proteins decides whether apoptosis will be induced or whether the cells will continue to survive. Extracellular growth factors inactivate Bad, thereby suppressing Bcl-2 production and thus protecting against apoptosis (12). Increased expression of this gene leads to the blocking of apoptosis, causing continuous overexpression of BCL2 and BCLX anti-apoptotic genes which is linked to poor prognosis and chemotherapy resistance (13). ...
Objectives:
In this study, we analyzed pTa bladder cancer (BC) for molecular markers BCL2, TP53, FOXA1, and GATA3 in relation to cancer recurrence.
Methods:
We analyzed samples of 79 patients with the pTa stage of BC using a real-time polymerase chain reaction (real-time PCR) between September 2018 and September 2020. The expression levels of BCL2, TP53, FOXA1, and GATA3 were compared with homologous non-tumor bladder tissue.
Results:
Expression of FOXA1, GATA3, and TP53 was significantly higher (p<0.01) in NMIBC samples compared to homologous non-tumor tissue. The expression of TP53 and FOXA1 in pTa was significantly lower (p<0.01) in the high-grade (HG) tumor when compared to the low-grade (LG) tumor. In contrast, the relative quantification (RQ) of GATA3 was significantly higher (p<0.01) in HG pTa. Patients with recurrence (pTa=33) had significantly higher expression of TP53, and GATA3 (p<0.01), and the gene of FOXA1 (p<0.01) had a significantly lower expression when compared to pTa tumors without recurrence. The expression of Bcl-2 was not statistically significant.
Conclusion:
Our results, indicate, that comparing expression levels of these genes in cancer and cancer-free tissue could provide valuable data, as patients with pTa BC recurrence within up to 54 months of follow-up had a significantly higher RQ of TP53, GATA3, and FOXA1 when compared to pTa BC patients without recurrence (Tab. 2, Fig. 8, Ref. 54). Text in PDF www.elis.sk Keywords: bladder cancer, gene expression, recurrence, GATA3, FOXA1, TP53, BCL2.
... Because clusterin has been found to be upregulated in multiple human cancers including prostate and breast cancers 1,2,6 , we were interested in whether the inactivation of Bax by clusterin might have a role in oncogenic transformation or tumour development. As Bax has a critical role in c-Myc-mediated apoptosis and transformation [18][19][20][21][22] , a well-established inducible Rat-1/c-MycER cell model system was utilized 22,23 . In this model, c-Myc polypeptide is fused with a portion of the human ...
... Reciprocally, increasing amounts of Bax were co-immunoprecipitated by anti-clusterin antibodies following c-Myc activation (Fig. 3e). Previously, either the elevated level of Bcl-2 anti-apoptotic proteins or the loss of Bax has been shown to cooperate with c-Myc in oncogenesis 18,22 . An assessment was made as to whether clusterin collaborated with c-Myc in oncogenic transformation. ...
... The protein product of Bcl-2 inhibits apoptosis by preserving the mitochondrial membrane integrity [15]. The increased expression of the anti-apoptotic Bcl-2 favors the accumulation of new mutations because it makes the removal of the genetically modified cell difficult, which can result in the appearance of cells with a malignant phenotype [16]. ...
... Polverini and Nor suggested that the upregulation of Bcl-2 in human premalignant and malignant keratinocytes may be an early key event in epithelial carcinogenesis [18]. In oral cancer, Bcl-2 is 3A 3B altered cell survival, thereby increasing the possibility of appearance and accumulations of mutations which provide a selective growth advantage to the neoplastic clone of cells comprising an oncogene or a tumor suppressor gene [16]. Bcl-2 expression was found to be increasing with increasing grades of dysplasia, thereby suggesting a possible role in malignant transformation [1, 19,21]. ...
... Back in 1984, Tsujimoto and colleagues found a chromosome translocation (t(14;18)) that linked the immunoglobulin heavy chain locus to a bcl-2 gene located on chromosome 18 [50]. Later it has been shown that the expression of bcl-2 promotes haemopoietic cell survival and provides modest growth advantage [51]. In addition, bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation [52]. ...
... The Bcl-2 family 'initiators' are pro-apoptotic members, often referred to as 'BH3-only proteins' because most members possess only the conserved BH3 domain [43,44]. Bcl2 is an inhibitory protein of apoptosis and benefits cell survival in the absence of cytokines [45]. Bax, the guardian member of the Bcl-2 family, is often referred to as a pro-survival protein because its overexpression inhibits apoptotic cell death. ...
Breast cancer is one of the leading causes of cancer mortality worldwide, and triple-negative breast cancer (TNBC) is the most problematic subtype. There is an urgent need to develop novel drug candidates for TNBC. Marine toxins are a valuable source for drug discovery. We previously identified αO-conotoxin GeXIVA[1,2] from Conus generalis, which is a selective antagonist of α9 nicotinic acetylcholine receptors (nAChRs). Recent studies indicated that α9 nAChR expression is positively correlated with breast cancer development; thus, α9 nAChR could serve as a therapeutic target for breast cancer. In this study, we aimed to investigate the in vivo antitumor effects of GeXIVA[1,2] on TNBC and to elucidate its underlying anticancer mechanism. Our data showed that GeXIVA[1,2] effectively suppressed 4T1 tumor growth in vivo at a very low dose of 0.1 nmol per mouse. Our results uncovered that the antitumor mechanism of GeXIVA[1,2] simultaneously induced apoptosis and blocked proliferation. Further investigations revealed that GeXIVA[1,2]-induced Caspase-3-dependent apoptosis was achieved through regulating Bax/Bcl-2 balance, and GeXIVA[1,2]-inhibited proliferation was mediated by the downregulation of the AKT-mTOR, STAT3 and NF-κB signaling pathways. Our study provides valuable arguments to demonstrate the potential of GeXIVA[1,2] as a novel marine-derived anticancer drug candidate for the treatment of TNBC.
... Under the activation of proapoptotic BH3-only proteins, BAX and BAK oligomerizes on mitochondrial outer membrane to form pores followed by release of cytochrome c and downstream activation of caspases (99). The discovery of BCL-2 as oncogene which had anti-apoptotic activities, its binding with BH3 only pro-apoptotic proteins, and further reports of evidence showing the selection of malignant cells with high BCL-2 expression has led to the stimulus for targeting BCL2 via small molecule inhibitors such as BH3 mimetics (example venetoclax) (100,101). ...
Acute myeloid leukemia (AML) is clinically and genetically a heterogeneous disease characterized by clonal expansion of abnormal hematopoietic progenitors. Genomic approaches to precision medicine have been implemented to direct targeted therapy for subgroups of AML patients, for instance, IDH inhibitors for IDH1/2 mutated patients, and FLT3 inhibitors with FLT3 mutated patients. While next generation sequencing for genetic mutations has improved treatment outcomes, only a fraction of AML patients benefit due to the low prevalence of actionable targets. In recent years, the adoption of newer functional technologies for quantitative phenotypic analysis and patient-derived avatar models has strengthened the potential for generalized functional precision medicine approach. However, functional approach requires robust standardization for multiple variables such as functional parameters, time of drug exposure and drug concentration for making in vitro predictions. In this review, we first summarize genomic and functional therapeutic biomarkers adopted for AML therapy, followed by challenges associated with these approaches, and finally, the future strategies to enhance the implementation of precision medicine.
... BCL-2 is overexpressed in lymphoid malignancies, and it is the predominant survival factor in many of the related tumor types 10 . Meanwhile overexpression of BCL-X L has been found correlated with drug resistance and disease progression of multiple solid tumors and hematologic malignancies 11,12 . ...
Overexpressed pro-survival B-cell lymphoma-2 (BCL-2) family proteins BCL-2 and BCL-XL can render tumor cells malignant. Leukemia drug venetoclax is currently the only approved selective BCL-2 inhibitor. However, its application has led to an emergence of resistant mutations, calling for drugs with an innovative mechanism of action. Herein we present cyclic peptides (CPs) with nanomolar-level binding affinities to BCL-2 or BCL-XL, and further reveal the structural and functional mechanisms of how these CPs target two proteins in a fashion that is remarkably different from traditional small-molecule inhibitors. In addition, these CPs can bind to the venetoclax-resistant clinical BCL-2 mutants with similar affinities as to the wild-type protein. Furthermore, we identify a single-residue discrepancy between BCL-2 D111 and BCL-XL A104 as a molecular “switch” that can differently engage CPs. Our study suggests that CPs may inhibit BCL-2 or BCL-XL by delicately modulating protein-protein interactions, potentially benefiting the development of next-generation therapeutics.
... A significant increase was observed in the expression level of BAX gene, a gene with a pro-apoptotic role in the apoptosis mechanism [12], at a dose of 5 M compared to the control group. A significant decrease was observed in the expression level of the BCL2 gene, a gene with an anti-apoptotic role in the apoptosis mechanism [13], at a dose of 5 M compared to the control group. A significant decrease was observed in the expression level of the MMP8 gene, a gene that has a role in the proliferation mechanism [14], at a dose of 5 M compared to the control group. ...
... Combination therapy of PDT with 5-ADC in vivo provides circumstantial evidence that there is a possible improved host immune response with combination therapy. BCL3 mediated repression of transcription is associated with the development and activation of immune cells (73). BCL2 on the other hand is recognized as an anti-apoptotic molecule, noted to be upregulated in tumour cells (74). ...
Introduction
Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2’-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood.
Methods
The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients.
Results
Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease.
Discussion
To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.
... Furthermore, it has anti-apoptotic effects that inhibit both perforin-and granzyme-mediated cell death [146]. In malignancies, BCL-2 proteins are often overexpressed on tumor cells and this has been identified as a key factor rendering tumor cells resistant to apoptosis [147]. These findings align with the finding that in cells from PLWH on ART containing inducible HIV BCL-2 was also expressed at a higher level [148]. ...
Introduction:
The main barrier to finding a cure against HIV is the latent HIV reservoir, which persists in people living with HIV (PLWH) despite antiretroviral treatment (ART). Here, we discuss recent findings from interventional studies using mono- and combination therapies aimed at enhancing immune-mediated killing of the virus with or without activating HIV from latency.
Areas covered:
We discuss latency reversal agents (LRAs), broadly neutralizing antibodies, immunomodulatory therapies, and studies aimed at inducing apoptosis.
Expert opinion:
The landscape of clinical trials for HIV cure and remission has evolved considerably over the past 10 years. Several novel interventions such as immune checkpoint inhibitors, therapeutic vaccines, and broadly neutralizing antibodies have been tested either alone or in combination with LRAs but studies have so far not shown a meaningful impact on the frequency of latently infected cells. Immunomodulatory therapies could work differently in the setting of antigen expression, that is, during active viremia, and timing of interventions could therefore, be key to future therapeutic success. Lessons learned from clinical trials aimed at HIV cure indicate that while we are still far from reaching a complete eradication cure of HIV, clinical interventions capable of inducing enhanced control of HIV replication in the absence of ART might be a more feasible goal.
... The BCL-2 protein family splits into three branches: pro-survival proteins, pro-apoptotic proteins and BH3-only pro-apoptotic proteins. BCL-2, among others, is a pro-survival protein of the mitochondrial signaling pathway [26]. The apoptosis-promoting proteins are the BCL-2-associated X protein (BAX), BCL-2 antagonist killer 1 (BAK) and perhaps BCL-2-related ovarian killer protein (BOK). ...
Osteoarthritis (OA) is one of the most common chronic diseases in human and animal joints. The joints undergo several morphological and histological changes during the development of radiographically visible osteoarthritis. The most discussed changes include synovial inflammation, the massive destruction of articular cartilage and ongoing joint destruction accompanied by massive joint pain in the later stadium. Either the increased apoptosis of chondrocytes or the insufficient apoptosis of inflammatory macrophages and synovial fibroblasts are likely to underly this process. In this review, we discuss the current state of research on the pathogenesis of OA with special regard to the involvement of apoptosis.
... Patients have a poor prognosis due to severe side effects and acquired drug resistance [4]. Overexpression of B-cell lymphoma 2 (Bcl-2) is a clinical feature of human hematologic malignancies that exacerbates the malignant state and drives the apoptosis resistance phenotype [5,6]. The identification of Bcl-2 as a therapeutic target ushered in a paradigm shift in the treatment of AML [7]. ...
Background
The identifying of B-cell lymphoma 2 (Bcl-2) as a therapeutic target has led to a paradigm shift in acute myeloid leukemia (AML) treatment. Pyroptosis is a novel antitumor therapeutic mechanism due to its cytotoxic and immunogenic effects. The combination of venetoclax and hypomethylating agents (HMAs) has been shown to lead to durable responses and significantly improve prognosis in patients with AML. However, our understanding of the mechanisms underlying this combinatorial activity is evolving.
Methods
We investigated whether the Bcl-2 inhibitor venetoclax induces AML cell pyroptosis and identified pyroptosis effector proteins. Via using western blotting, immunoprecipitation, RNA interference, CCK8 assays, and LDH assays, we explored the mechanism underlying the pyroptotic effect. The relationship between the expression of the pyroptosis effector protein GSDME and AML prognosis was investigated. The effect of GSDME demethylation combined with venetoclax treatment on pyroptosis was investigated and confirmed in mouse models and clinical samples.
Results
Venetoclax induces pyroptosis that is mediated by caspase-3-dependent GSDME cleavage. Mechanistically, venetoclax upregulates caspase-3 and GSDME cleavage by activating the intrinsic apoptotic pathway. GSDME is downregulated in AML by promoter methylation, and low GSDME expression is significantly associated with poor prognosis, based on public databases and patient sample analysis. In vivo and in vitro experiments showed that GSDME overexpression or HMAs-mediated restoration of GSDME expression significantly increased venetoclax-induced pyroptosis in AML.
Conclusion
GSDME-mediated pyroptosis may be a novel aspect of the antileukemic effect of Bcl-2 inhibitors. This finding offers new insights into potential biomarkers and therapeutic strategies, identifying an important mechanism explaining the clinical activity of venetoclax and HMAs in AML.
... Expression of Bcl-2 protein is involved in hindering the cell death, following multiple pathological and physiological stimuli (Vaux et al. 1988;McDonnell et al. 1989). Bcl-2 proteins are confined to perinuclear membrane, SER (smooth endoplasmic reticulum), and mitochondria (Krajewski et al. 1993;Hockenbery et al. 1990). ...
Protein–protein interactions (PPIs) have been sought as putative therapeutic targets for the advancement of various new treatments. This chapter deals with the various studies that have successfully discovered small-molecule inhibitors (SMIs) associated with particular disease-causing PPI. The employed methodologies in these studies as well as the conclusive results have been thoroughly discussed. Further, other aspects of the discovery like optimization of the process, strategizing drug binding, selection of targets have also been delineated. This chapter thus provides the reader with a comprehensive account of the current state-of-art in the discovery of small molecules inhibiting PPIs. It also throws light on the future potential of these small molecules as commercial drug candidates.
... survival of cells harboring harmful DNA damage. 47 BCL2 itself, the prototypic oncogene that operates by promoting cell survival, 48 can prevent apoptosis, along with other anti-apoptotic members of its family, in response to numerous stresses to which pre-malignant or rapidly expanding malignant cells are exposed. These include genotoxic damage, oxidative and metabolic stresses, and limited growth factor availability. ...
Cancers are genetically driven, rogue tissues which generate dysfunctional, obdurate organs by hijacking normal, homeostatic programs. Apoptosis is an evolutionarily conserved regulated cell death program and a profoundly important homeostatic mechanism that is common (alongside tumor cell proliferation) in actively growing cancers, as well as in tumors responding to cytotoxic anti‐cancer therapies. Although well known for its cell‐autonomous tumor‐suppressive qualities, apoptosis harbors pro‐oncogenic properties which are deployed through non‐cell‐autonomous mechanisms and which generally remain poorly defined. Here, the roles of apoptosis in tumor biology are reviewed, with particular focus on the secreted and fragmentation products of apoptotic tumor cells and their effects on tumor‐associated macrophages, key supportive cells in the aberrant homeostasis of the tumor microenvironment. Historical aspects of cell loss in tumor growth kinetics are considered and the impact (and potential impact) on tumor growth of apoptotic‐cell clearance (efferocytosis) as well as released soluble and extracellular vesicle‐associated factors are discussed from the perspectives of inflammation, tissue repair, and regeneration programs. An “apoptosis‐centric” view is proposed in which dying tumor cells provide an important platform for intricate intercellular communication networks in growing cancers. The perspective has implications for future research and for improving cancer diagnosis and therapy.
... Apoptosis results in DNA fragmentation and thus plays a substantial role in the disruption of gastric mucosal wall integrity [4,75]. However, the anti-apoptotic protein B cell lymphoma-2 (Bcl-2) regulates the apoptotic machinery by interacting with and neutralizing the mitochondrial proapoptotic proteins [76]. ROS have been implicated in the activation of mitochondrial apoptotic protein (Bax) and eventually result in the activation of executioner caspases [75,77]. ...
Gastric ulcer is a serious disease that affects millions of individuals worldwide. Alcohol consumption is a major contributor to the disease pathogenesis and ethanol-induced ulcer in rats closely recapitulates the clinical pathology of ulcer. In this study, rats were pretreated with carvacrol (CAR,50 and 100 mg/kg, orally) 1 h before absolute ethanol administration to induce gastric ulcer. CAR prevented ethanol-induced increases in gastric volume and acidity while restored mucin content. The gastro-protective activity of CAR, particularly the higher dose (100 mg/kg), was further supported by histopathological examination, as manifested by reduced gastric lesions. Interestingly, oxidative stress is linked to early stages of ulcer development and progression. In this study, ethanol administration upregulated the levels of ROS-producing enzymes, NADPH oxidase homologs 1 and 4 (Nox1 and Nox4) and lipid peroxides while depleting the antioxidant defense mechanisms, including GSH, Glutathione Peroxidase (GPX) and catalase. Interestingly, these alterations were significantly ameliorated by CAR pretreatment. Additionally, CAR possesses anti-inflammatory and anti-apoptotic activities. Pretreatment with CAR blunted ethanol-induced increases in inflammatory cytokines (NF-κB and TNF-α) and rectified the apoptosis regulator (Bax/Bcl2 ratio) in gastric tissue. Moreover, the docking simulation of CAR illustrated good fitting and interactions with GPX, Nox1 and TNF-α through the formation of hydrogen and hydrophobic (pi-H) bonds with conservative amino acids, thus, further supporting the anti-inflammatory and antioxidant effects underlying the gastroprotective effects of CAR. In conclusion, this study elucidates, using in silico and in vivo models, that the gastroprotective activity of CAR is attributed, at least in part, to its mucin-secretagogue, antioxidative, anti-inflammatory, and anti-apoptotic mechanisms.
... Overexpression of Bcl2 is common in many types of human cancer and is highly correlated with therapy resistance. In general, Bcl2 overexpression does not promote cell proliferation, but rather, overexpression of Bcl2 inhibits cell death via evading apoptotic checkpoint [39]. Cancer cells with a high level of Bcl2 have unbalanced apoptotic regulators and are addicted to Bcl2. ...
Colorectal cancer (CRC) driven by PTEN deficiency exhibits high risk of metastasis, advancement of tumor stages and chemotherapy resistance, where no effective therapy has been developed. In this study, we performed a synthetic lethal drug screening in CRC and found that PTEN-deficient CRC cells are highly vulnerable to MDM2 inhibition. MDM2 inhibitor treatment or its silencing selectively inhibited the growth of PTEN-deficient CRC in vitro and in mice models. Mechanistically, PTEN loss increased the level of active AKT and subsequently increased MDM2 phosphorylation, thereby limiting the p53 functions in PTEN-/- CRC cells. MDM2 inhibition in turn activated p53 in CRC, particularly in PTEN-/- CRC cells. The synthetic lethal effect of MDM2 inhibitor was largely dependent on p53, because p53 silenced cells or cells lacking p53 failed to exhibit synthetic lethality in PTEN-deficient cells. We further showed that MDM2 inhibition led to the p53-dependent reversal of Bcl2-Bax ratio, which contributed to mitochondria-mediated apoptotic cell death in PTEN-deficient CRC. This study suggests that pharmacological targeting of MDM2 could be a potential therapeutic strategy for PTEN-deficient CRC.
... In such instances, there is minimal variability in how the fusion occurs, and they are frequently recognized as recurrent oncofusions in large-scale studies. For example, the oncogenic potential of the IGH::BCL2 oncofusion is derived from the combination of the anti-apoptotic BCL2 with the highly expressed immunoglobulin locus of IGH [50,51]. A similar mechanism is often present in prostate cancer, where the TMPRSS2::ERG oncofusion is found in roughly 50% of tumors and MAN2A1::FER, MTOR::TP52BP1, and SLC45A2::AMACR occur at lower frequencies. ...
Simple Summary
Oncofusions, or cancer-associated fusion mutations, are driving forces in cancer development. Advanced sequencing technologies have revolutionized their identification, opening new avenues in cancer research. Oncofusions manipulate cellular signaling pathways and show promise as targets for therapy and diagnostic markers. Further research is needed to understand their functional impact and harness their potential for precision cancer treatment.
Abstract
Cancer-associated gene fusions, also known as oncofusions, have emerged as influential drivers of oncogenesis across a diverse range of cancer types. These genetic events occur via chromosomal translocations, deletions, and inversions, leading to the fusion of previously separate genes. Due to the drastic nature of these mutations, they often result in profound alterations of cellular behavior. The identification of oncofusions has revolutionized cancer research, with advancements in sequencing technologies facilitating the discovery of novel fusion events at an accelerated pace. Oncofusions exert their effects through the manipulation of critical cellular signaling pathways that regulate processes such as proliferation, differentiation, and survival. Extensive investigations have been conducted to understand the roles of oncofusions in solid tumors, leukemias, and lymphomas. Large-scale initiatives, including the Cancer Genome Atlas, have played a pivotal role in unraveling the landscape of oncofusions by characterizing a vast number of cancer samples across different tumor types. While validating the functional relevance of oncofusions remains a challenge, even non-driver mutations can hold significance in cancer treatment. Oncofusions have demonstrated potential value in the context of immunotherapy through the production of neoantigens. Their clinical importance has been observed in both treatment and diagnostic settings, with specific fusion events serving as therapeutic targets or diagnostic markers. However, despite the progress made, there is still considerable untapped potential within the field of oncofusions. Further research and validation efforts are necessary to understand their effects on a functional basis and to exploit the new targeted treatment avenues offered by oncofusions. Through further functional and clinical studies, oncofusions will enable the advancement of precision medicine and the drive towards more effective and specific treatments for cancer patients.
... Follicular lymphoma bears many chromosomal aberrations that vary in number, mostly of unknown or questionable contribution to pathogenesis. 9,10 The t(14;18)(q32;q21) translocation was first detected by karyotypic analysis, which is at present not used for this purpose. 11 A commonly used method for translocation detection is Flourescence In Situ Hybridisation (FISH). ...
Background:
t(14;18)(q32;q21) translocation is an important genetic feature of follicular lymphoma resulting in antiapoptotic B-cell lymphoma 2 (BCL2) protein overexpression. On chromosome 18 breakpoint-site variation is high but does not affect BCL2. Breakpoint most commonly occurs at major breakpoint region (MBR) but may happen at minor cluster region (mcr) and between MBR and mcr at 3'MBR and 5'mcr. The aim of this study was to analyze the correlation of t(14;18)(q32;q21) breakpoint site with clinical characteristics in follicular lymphoma.
Patients and methods:
We included patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treatment and had the t(14;18)(q32;q21) translocation detected by polymerase chain reaction (PCR) at MBR, mcr or 3'MBR prior to first treatment. Among patients with different breakpoints, sex, age, disease grade, stage, B-symptoms, follicular lymphoma international prognostic index (FLIPI), presence of bulky disease, progression free survival and overall survival were compared.
Results:
Of 84 patients, 63 had breakpoint at MBR, 17 at mcr and 4 at 3'MBR. At diagnosis, the MBR group had a significantly lower disease stage than the mcr group. Although not significant, in the MBR group we found a higher progression-free survival (PFS) and overall survival (OS), lower grade, age, FLIPI, and less B-symptoms.
Conclusions:
Compared to patients with mcr breakpoint, those with MBR breakpoint seem to be characterised by more favourable clinical characteristics. However, a larger study would be required to support our observation.
... The B-cell leukaemia-2 gene (Bcl-2) is a proto-oncogene that inhibits apoptosis (VAUX et al., 1988). In our study, the highest levels of expression were found in mucinous cystadenoma (O9T) and serous adenocarcinomas (OO2T, O11T) meaning that in these tumors apoptosis was inhibited. ...
There is a wide group of ovarian tumors, malignant as well as benign. Histopathological examination is used as a primary source of identifying the difference between malignant and benign processes. We analyzed ovarian tissue samples of twelve women, ages ranging from 21 to 77. Samples of 8 benign, 1 borderline, and 3 malignant ovarian tumors were included in the study. Using the quantitative PCR method, genes indicating apoptotic processes in tissue cells were evaluated, based on monitoring their relative expression. Relative gene expression was monitored for anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax, and effector protein Cas3. Expression of gene for protein Bax and Bcl-2 is increased and Bax predominates, which indicates that the initiation of apoptosis has begun, mostly in benign tumors. Bcl-2 was elevated in samples of borderline and malignant tumors while Bax was decreased, which indicates the inhibition of apoptosis in these samples.
... It was the culmination of a 25-year international research effort that originated with the elucidation of BCL2's prosurvival function in 1988. 5,6 VEN was derived from the first true BH3-mimetic, ABT-737, a tool compound that inhibited all of BCL2, BCL-X L , and BCL-W. 7 Targeting BCL-X L , however, caused acute-onset thrombocytopenia in vivo, linked to its crucial role in regulating platelet lifespan. ...
Altering the natural history of acute myeloid leukemia (AML) in unfit and older patients has proved a highly challenging hurdle, despite several decades of concerted clinical trial effort. The arrival of venetoclax (VEN) to the clinical stage represents the most important therapeutic advance to date for older patients with AML. In this review, we will explain how and why VEN works, summarize its remarkable pathway to regulatory approval, and highlight the key milestones that have been important for its successful development in AML. We also provide perspectives on some of the challenges associated with using VEN in the clinic, emerging knowledge regarding mechanisms of treatment failure, and current clinical research directions likely to shape how this drug and others in this new class of anticancer agents are used in the future.
... In this assessment, only compound 11 displays an interesting profile; indeed, two genes were less expressed as compared with control conditions, namely, Bcl-2 and mTOR. The former one has an anti-apoptosis function [38][39][40] and its depressed levels may be associated with a smaller cellular proliferation [41], but the inhibitory effect induced by compound 11 was not statistically significant as compared with control conditions (B2M levels). ...
A series of 2-phenylamino-3-acyl-1,4-naphtoquinones were evaluated regarding their in vitro antiproliferative activities using DU-145, MCF-7 and T24 cancer cells. Such activities were discussed in terms of molecular descriptors such as half-wave potentials, hydrophobicity and molar refractivity. Compounds 4 and 11 displayed the highest antiproliferative activity against the three cancer cells and were therefore further investigated. The in silico prediction of drug likeness, using pkCSM and SwissADME explorer online, shows that compound 11 is a suitable lead molecule to be developed. Moreover, the expressions of key genes were studied in DU-145 cancer cells. They include genes involved in apoptosis (Bcl-2), tumor metabolism regulation (mTOR), redox homeostasis (GSR), cell cycle regulation (CDC25A), cell cycle progression (TP53), epigenetic (HDAC4), cell-cell communication (CCN2) and inflammatory pathways (TNF). Compound 11 displays an interesting profile because among these genes, mTOR was significantly less expressed as compared to control conditions. Molecular docking shows that compound 11 has good affinity with mTOR, unraveling a potential inhibitory effect on this protein. Due to the key role of mTOR on tumor metabolism, we suggest that impaired DU-145 cells proliferation by compound 11 is caused by a reduced mTOR expression (less mTOR protein) and inhibitory activity on mTOR protein.
... Therefore, forced activation of low-density lipoprotein receptorassociated protein 6 signaling may accelerate the generation of functional vessels (Silva et al., 2017). Hypoxia causes the stable expression of hypoxia inducible factor 1 subunit alpha (HIF-1α) and upregulation of VEGF (Pugh and Ratcliffe, 2003), promoting endothelial differentiation of SHEDs (Han Y. et al., 2022), while BCL2 apoptosis regulator (BCL2) is an anti-apoptotic gene that can inhibit cell apoptosis and prolong cell life (Vaux et al., 1988). Overexpression of BCL2 and hypoxic preconditioning could enhance cell survival and angiogenesis after DPSC implantation (Dissanayaka et al., 2020). ...
One of the difficulties of pulp regeneration is the rapid vascularization of transplanted engineered tissue, which is crucial for the initial survival of the graft and subsequent pulp regeneration. At present, prevascularization techniques, as emerging techniques in the field of pulp regeneration, has been proposed to solve this challenge and have broad application prospects. In these techniques, endothelial cells and pericytes are cocultured to induce intercellular communication, and the cell coculture is then introduced into the customized artificial vascular bed or induced to self-assembly to simulate the interaction between cells and extracellular matrix, which would result in construction of a prevascularization system, preformation of a functional capillary network, and rapid reconstruction of a sufficient blood supply in engineered tissue after transplantation. However, prevascularization techniques for pulp regeneration remain in their infancy, and there remain unresolved problems regarding cell sources, intercellular communication and the construction of prevascularization systems. This review focuses on the recent advances in the application of prevascularization techniques for pulp regeneration, considers dental stem cells as a potential cell source of endothelial cells and pericytes, discusses strategies for their directional differentiation, sketches the mechanism of intercellular communication and the potential application of communication mediators, and summarizes construction strategies for prevascularized systems. We also provide novel ideas for the extensive application and follow-up development of prevascularization techniques for dental pulp regeneration.
In 2024, the World Health Organization (WHO) launched a new classification of lymphoid neoplasms, a revision of the previously used Revised 4th Edition of their classification (WHO-4R). However, this means that two classifications are now in simultaneous use: the 5th Edition of the WHO classification (WHO-5) and the International Consensus Classification (ICC). Instead of a comprehensive review of each disease entity, as already described elsewhere, this review focuses on revisions made in both the WHO-5 and ICC from WHO-4R and discrepancies between them regarding B-cell neoplasms. Similarities include cutaneous marginal zone lymphoma, cold agglutinin disease, non-primary effusion lymphoma-type effusion-based lymphoma, and gray zone lymphoma. Differences include plasma cell neoplasms, high-grade B-cell lymphoma (double hit lymphoma), follicular lymphoma, LPD with immune deficiency and dysregulation, extranodal large B-cell lymphoma, transformations of indolent B-cell lymphomas, and diffuse large B-cell lymphoma, not otherwise specified. Understanding the similarities and differences between the two latest classifications will aid daily diagnostic practice and future research on lymphoid neoplasms.
The approval of venetoclax, a B-cell lymphoma-2 (Bcl-2) selective inhibitor, for the treatment of chronic lymphocytic leukemia demonstrated that the antiapoptotic protein Bcl-2 is a druggable target for B-cell malignancies. However, venetoclax’s limited potency cannot produce a strong, durable clinical benefit in other Bcl-2-mediated malignancies (e.g., diffuse large B-cell lymphomas) and multiple recurrent Bcl-2 mutations (e.g., G101V) have been reported to mediate resistance to venetoclax after long-term treatment. Herein, we described novel Bcl-2 inhibitors with increased potency for both wild-type (WT) and mutant Bcl-2. Comprehensive structure optimization led to the clinical candidate BGB-11417 (compound 12e, sonrotoclax), which exhibits strong in vitro and in vivo inhibitory activity against both WT Bcl-2 and the G101V mutant, as well as excellent selectivity over Bcl-xL without obvious cytochrome P450 inhibition. Currently, BGB-11417 is undergoing phase II/III clinical assessments as monotherapy and combination treatment.
The aim of this study was to determine whether endotoxin, hypoxia, and prematurity are risk factors for developing necrotizing enterocolitis (NEC), which was achieved by investigating the pathogenesis of necrotizing enterocolitis in rats. To this end, we used premature Sprague-Dawley (SD) rat pups delivered by cesarean section at a gestational age of 21 days (Pre-term group) as well as Full-term SD rat pups four days after birth (Full-term group). The pups were exposed to lipopolysaccharides (LPS) and hypoxia to induce necrotizing enterocolitis. It was seen that both Full- and Pre-term rats were resulted in necrotizing enterocolitis. The results indicated that, compared to the control groups, the degree of apoptosis was elevated in both the Pre-term and Full-term NEC rats. The Full-term group also showed a reduction in Bcl-2 levels and an elevation in the ratio of Bax-to-Bcl-2 levels compared to the control group. Meanwhile, the Pre-term group showed a significantly increased expression level of RIPK1, which implies the induction of RIPK1-dependent apoptosis. This suggests that the pathophysiology of necrotizing enterocolitis induced by LPS+ hypoxia is associated with programmed cell death pathway. It appears that the apoptotic pathway of the Bax/Bcl-2 system is the main mechanism in Full-term rats. By contrast, a number of other mechanisms, including TNF-induced apoptosis mechanism, may work together for necrotizing enterocolitis development in Pre-term rats. Further studies are needed to elucidate the different pathogenesis of necrotizing enterocolitis development between Pre-term and Full-term rats.
Regulated cell death mediated by dedicated molecular machines, known as programmed cell death, plays important roles in health and disease. Apoptosis, necroptosis and pyroptosis are three such programmed cell death modalities. The caspase family of cysteine proteases serve as key regulators of programmed cell death. During apoptosis, a cascade of caspase activation mediates signal transduction and cellular destruction, whereas pyroptosis occurs when activated caspases cleave gasdermins, which can then form pores in the plasma membrane. Necroptosis, a form of caspase-independent programmed necrosis mediated by RIPK3 and MLKL, is inhibited by caspase-8-mediated cleavage of RIPK1. Disruption of cellular homeostatic mechanisms that are essential for cell survival, such as normal ionic and redox balance and lysosomal flux, can also induce cell death without invoking programmed cell death mechanisms. Excitotoxicity, ferroptosis and lysosomal cell death are examples of such cell death modes. In this Review, we provide an overview of the major cell death mechanisms, highlighting the latest insights into their complex regulation and execution, and their relevance to human diseases.
Pancreatic cancer is the seventh leading cause of cancer-related death worldwide, and despite advancements in disease management, the 5-year survival rate stands at only 12%. Triptolides have potent anti-tumor activity against different types of cancers, including pancreatic cancer, however poor solubility and toxicity limit their translation into clinical use. We synthesized a novel pro-drug of triptolide, ( E )-19-[(1'-benzoyloxy-1'-phenyl)-methylidene]-Triptolide (CK21), which was formulated into an emulsion for in vitro and in vivo testing in rats and mice, and using human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids. A time-course transcriptomic profiling of tumor organoids treated with CK21 in vitro was conducted to define its mechanism of action, as well as transcriptomic profiling at a single time point post-CK21 administration in vivo. Intravenous administration of emulsified CK21 resulted in the stable release of triptolide, and potent anti-proliferative effects on human pancreatic cancer cell lines and patient-derived pancreatic tumor organoids in vitro, and with minimal toxicity in vivo . Time course transcriptomic profiling of tumor organoids treated with CK21 in vitro revealed <10 differentially expressed genes (DEGs) at 3 h and ~8,000 DEGs at 12 h. Overall inhibition of general RNA transcription was observed, and Ingenuity pathway analysis together with functional cellular assays confirmed inhibition of the NF-κB pathway, increased oxidative phosphorylation and mitochondrial dysfunction, leading ultimately to increased reactive oxygen species (ROS) production, reduced B-cell-lymphoma protein 2 (BCL2) expression, and mitochondrial-mediated tumor cell apoptosis. CK21 is a novel pro-drug of triptolide that exerts potent anti-proliferative effects on human pancreatic tumors by inhibiting the NF-κB pathway, leading ultimately to mitochondrial-mediated tumor cell apoptosis.
Extracellular vesicles (EVs) are small lipid bilayer–enclosed vesicles that mediate vital cellular communication by transferring cargo between cells. Among these, tissue-derived extracellular vesicles (Ti-EVs) stand out due to their origin from the tissue microenvironment, providing a more accurate reflection of changes in this setting. This unique advantage makes Ti-EVs valuable in investigating the intricate relationship between extracellular vesicles and cancer progression. Despite considerable research efforts exploring the association between Ti-EVs and cancers, a comprehensive clustering or grouping of these studies remains lacking. In this review, we aim to fill this gap by presenting a comprehensive synthesis of the mechanisms underlying Ti-EV generation, release, and transport within cancer tissues. Moreover, we delve into the pivotal roles that Ti-EVs play in cancer progression, shedding light on their potential as diagnostic and therapeutic tools. The review culminates in the construction of a comprehensive functional spectrum of Ti-EVs, providing a valuable reference for future research endeavors. By summarizing the current state of knowledge on Ti-EVs and their significance in tumor biology, this work contributes to a deeper understanding of cancer microenvironment dynamics and opens up avenues for harnessing Ti-EVs in diagnostic and therapeutic applications.
Sepsis-induced cardiac dysfunction represents a major cause of high mortality in intensive care units with limited therapeutic options. Golgi protein 73 (GP73) has been implicated in various diseases. However, the role of GP73 in LPS-induced cardiac dysfunction is unclear. Here, we established sepsis-induced cardiac dysfunction model by LPS administration in wild type (WT) and GP73 knockout ( GP73 -/- ) mice. We found GP73 was increased in LPS-treated mouse hearts and LPS-cultured neonatal rat cardiomyocytes (NRCMs). Knockout of GP73 alleviated myocardial injury and improved cardiac dysfunction. Moreover, depletion of GP73 in NRCMs relieved LPS-induced cardiomyocyte apoptosis and activated myocardial autophagy. Therefore, GP73 is a negative regulator in LPS-induced cardiac dysfunction by promoting cardiomyocyte apoptosis and inhibiting cardiomyocyte autophagy.
The B cell lymphoma-2 (BCL-2) family of proteins plays a critical role in the intrinsic pathway of apoptosis. It is therefore not surprising that this pathway is frequently dysregulated in numerous malignancies, including acute myeloid leukemia (AML), in order to evade apoptosis. In the last 25 years, research into the pathobiology of AML has focused intensely on the antiapoptotic proteins, BCL-2, and myeloid cell leukemia-1 (MCL-1), whose overexpressions are associated with enhanced survival and chemoresistance of leukemic cells. In light of this, BCL-2 and MCL-1 have been attractive targets in the development of novel agents to treat AML. Many BCL-2 and MCL-1 inhibitors have yielded promising results in preclinical trials and are currently undergoing evaluation in clinical trials. Recently, venetoclax, a first-in-class selective oral BCL-2 inhibitor, was approved for upfront treatment of AML in the unfit or elderly population and had revolutionized the therapeutic landscape of AML. In this chapter, we will review the role of BCL-2 and MCL-1 in AML as well as the preclinical and clinical data supporting the use of BCL-2 and MCL-1 inhibitors in AML treatment. Furthermore, we will discuss the mechanisms of resistance to BCL-2 inhibitors and highlight ongoing clinical trials of combination therapies aimed at overcoming such resistance pathways.
Programmed cell death (self-induced) is intrinsic to all cellular life forms, including unicellular organisms. However, cell death research has focused on animal models to understand cancer, degenerative disorders, and developmental processes. Recently delineated suicidal death mechanisms in bacteria and fungi have revealed ancient origins of animal cell death that are intertwined with immune mechanisms, allaying earlier doubts that self-inflicted cell death pathways exist in microorganisms. Approximately 20 mammalian death pathways have been partially characterized over the last 35 years. By contrast, more than 100 death mechanisms have been identified in bacteria and a few fungi in recent years. However, cell death is nearly unstudied in most human pathogenic microbes that cause major public health burdens. Here, we consider how the current understanding of programmed cell death arose through animal studies and how recently uncovered microbial cell death mechanisms in fungi and bacteria resemble and differ from mechanisms of mammalian cell death.
Expected final online publication date for the Annual Review of Genetics, Volume 57 is November 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Background
The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy.
Methods
This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression.
Results
A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4–62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS.
Conclusion
In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.
In recent years, the therapeutic landscape of myeloid malignancies has been completely revolutionized by the introduction of several new drugs, targeting molecular alterations or pathways crucial for leukemia cells survival. Particularly, many agents targeting apoptosis have been investigated in both pre-clinical and clinical studies. For instance, venetoclax, a pro-apoptotic agent active on BCL-2 signaling, has been successfully used in the treatment of acute myeloid leukemia (AML). The impressive results achieved in this context have made the apoptotic pathway an attractive target also in other myeloid neoplasms, translating the experience of AML. Therefore, several drugs are now under investigation either as single or in combination strategies, due to their synergistic efficacy and capacity to overcome resistance. In this paper, we will review the mechanisms of apoptosis and the specific drugs currently used and under investigation for the treatment of myeloid neoplasia, identifying critical research necessities for the upcoming years.
Targeting the B-cell lymphoma 2 (Bcl-2) family proteins has been the backbone for hematological malignancies with overall survival improvements. The Bcl-2 family is a major player in apoptosis regulation and, has captured the researcher’s interest in the treatment of solid tumors. Sarcomas are a heterogeneous group of diseases, comprising several entities, with high morbidity and mortality and with few specific therapies available. The treatment for sarcomas is based on platinum regimens, with variable results and poor outcomes, especially in advanced lesions. The high number of different sarcoma entities makes treatment standardization as well as the performance of clinical trials difficult. The use of Bcl-2 family members modifiers has revealed promising results in in vitro and in vivo models and may be a valid option, especially when used in combination with chemotherapy. In this article, a revision of these results and possibilities for the use of Bcl-2 family members inhibitors in sarcomas was performed.
Phytochemical study of the ethyl acetate root extract of Zygophyllum album has resulted in the isolation of a new saponin, Zygo-albuside D (1), along with two known compounds; (3-O-[β-D-quinovopyranosyl]-quinovic acid) (2), which is first reported in the root, and catechin (3), first reported in the genus. Their chemical structures were established by NMR and high-resolution mass spectrometry (HRMS). The new saponin (1) exhibited promising cytotoxicity with IC50 values of 3.5 and 5.52 μM on A549 and PC-3 cancer cell lines, respectively, compared to doxorubicin with IC50 values of 9.44 and 11.39 μM on A549 and PC-3 cancer cell lines, respectively. While it had an IC50 value of 46.8 μM against WISH cells. Investigating apoptosis-induction, compound 1 induced total apoptotic cell death in A549 lung cancer cells by 32-fold; 21.53% compared to 0.67% in the untreated control cells. Finally, it upregulated the pro-apoptotic genes and downregulated the antiapoptotic gene using gene expression levels. Compound 1 exhibited remarkable CDK-2 target inhibition by 96.2% with an IC50 value of 117.6 nM compared to Roscovitine. The molecular docking study further confirmed the binding affinity of compound 1 as CDK2 and Bcl2 inhibitors that led to apoptosis induction in A549 cancer cells. Hence, this study highlights the importance of compound 1 in the design of a new anticancer agent with specific mechanisms.
The proteins of the BCL-2 family are key regulators of mitochondrial apoptosis, acting as either promoters or inhibitors of cell death. The functional interplay and balance between the opposing BCL-2 family members control permeabilization of the outer mitochondrial membrane, leading to the release of activators of the caspase cascade into the cytosol and ultimately resulting in cell death. Despite considerable research, our knowledge about the mechanisms of the BCL-2 family of proteins remains insufficient, which complicates cell fate predictions and does not allow us to fully exploit these proteins as targets for drug discovery. Detailed understanding of the formation and molecular architecture of the apoptotic pore in the outer mitochondrial membrane remains a holy grail in the field, but new studies allow us to begin constructing a structural model of its arrangement. Recent literature has also revealed unexpected activities for several BCL-2 family members that challenge established concepts of how they regulate mitochondrial permeabilization. In this Review, we revisit the most important advances in the field and integrate them into a new structure-function-based classification of the BCL-2 family members that intends to provide a comprehensive model for BCL-2 action in apoptosis. We close this Review by discussing the potential of drugging the BCL-2 family in diseases characterized by aberrant apoptosis.
Proteins are the workhorses of cells, performing most of the important functions which allow cells to use nutrients and grow, communicate among each other, and importantly, die if aberrant behavior is detected. How were proteins discovered? What is their role in cells? How do dysfunctional proteins give rise to cancers? Landmark Experiments in Protein Science explores the manner in which the inner workings of cells were elucidated, with a special emphasis on the role of proteins. Experiments are discussed in a manner as to understand what questions were being asked that prompted the experiments and what technical challenges were faced in the process; and results are presented and discussed using primary data and graphs.
Key Features:
- Describes landmark experiments in cell biology and biochemistry.
- Discusses the "How" and "Why" of historically important experiments.
- Includes primary, original data and graphs.
- Emphasizes biological techniques, that help understand how many of the experiments performed were possible.
- Documents, chronologically, how each result fed into the next experiments.
The most common translocation in human lymphoma, the t(14;18)(q32;q21), generates heterogeneous 4.2-7.2 kb Bcl-2-immunoglobulin (Ig) chimeric mRNAs resulting from alternative Bcl-2 5' exons and varied Ig 3' untranslated regions (UT). The normal human Bcl-2 gene has a three exon structure with an untranslated first exon, a facultative 220 bp intron I, but an enormous 370 kb intron II. S1 protection and primer extension analysis defined initiation sites in exon II associated with classic promoter elements and a decanucleotide (ATG-CAAAGCA) homologous with Ig variable region enhancers. Multiple initiation sites were also found in a GC-rich region with Sp1 binding motifs in exon I. Most t(14;18) breakpoints cluster within the 3' UT of Bcl-2 implicating that event in gene deregulation. The Bcl-2 gene introduced into the Ig constant (C gamma) locus of SU-DHL-6 displayed somatic mutation. While Bcl-2--Ig mRNAs demonstrated an unaltered 2.5 h half-life, the Bcl-2--Ig gene revealed an inappropriately high rate of transcription for a mature B-cell. This indicates the translocated Bcl-2 allele has escaped normal control mechanisms.
The normal cellular counterpart of the v-fms oncogene product is a receptor for the mononuclear phagocyte colony-stimulating
factor, CSF-1. An interleukin-3 (IL-3)-dependent mouse myeloid cell line, FDC-P1, was infected with a murine retrovirus vector
containing v-fms linked to a gene encoding resistance to neomycin (neo). Infected cells selected for resistance to the aminoglycoside
G418 contained few proviral DNA copies per haploid genome, expressed low levels of the v-fms-coded glycoprotein, remained
IL-3 dependent for growth, and were nontumorigenic in nude mice. In contrast, infected cells selected for their ability to
grow in the absence of IL-3 contained an increased number of proviral insertions, expressed high levels of the v-fms-coded
glycoprotein, and were tumorigenic in nude mice. The IL-3-independent cells expressed IL-3 receptors of comparable number
and affinity to those detected in uninfected FDC-P1 cells and did not produce a growth factor able to support replication
of the parental cells. Thus, the synthesis of high levels of the v-fms gene product in FDC-P1 cells abrogated their requirement
for IL-3 and rendered the cells tumorigenic by a nonautocrine mechanism. The data suggest that v-fms encodes a promiscuous
tyrosine kinase able to transform cells of the myeloid lineage that do not normally express CSF-1 receptors.
We examined the expression of the Bcl-2 gene at chromosome segment 18q21, that is translocated into the Ig heavy chain gene locus in t(14;18) bearing lymphomas. Bcl-2, while B cell associated, is expressed in a variety of hematopoietic lineages including T cells. Bcl-2 mRNA levels are high during pre-B cell development, the time at which the t(14;18) translocation occurs, but are down regulated with maturation. Like certain other oncogenes, Bcl-2 is quiescent in resting B cells but up-regulated with B cell activation. Mature B cell lymphomas with a t(14;18) have log-folds more mRNA than matched counterparts without the translocation. A sensitive S1 protection assay revealed that all transcripts in t(14;18) B cells were Bcl-2-Ig fusion mRNAs and originated from the translocated allele. Thus, there is a marked deregulation of Bcl-2 when it is introduced into the Ig locus in t(14;18) lymphomas.
Cell lines have been produced from long-term cultures of mouse bone marrow that require a factor, present in WEHI-3 conditioned medium (CM) or in spleen CM, for their sustained growth. The cell lines were obtained from nonvirus-treated cultures, are nonleukemic, maintain a normal karyotype, and form colonies showing granulocyte maturation when plated in soft agar. Granulocyte/macrophage (GM) colony-stimulating factor is not the inductive moiety involved in the maintenance of proliferation of these cells. It is suggested that the cell lines represent a self-renewing population of cells ancestral to GM colony-forming cells, which may be responding to a hitherto unrecognized regulator.
cDNAs for the bcl-2 mRNA were cloned from a human common acute lymphoblastic leukemia cell line. Nucleotide sequence analyses showed that the 6 kb bcl-2 mRNA potentially encodes a 26 kd protein that is homologous to a predicted Epstein-Barr virus protein. Most t(14;18) translocation breakpoints cluster within a narrow region of a 5.4 kb exon that contains a long 3'-untranslated region of the bcl-2 mRNA. As a result of t(14;18) translocation, hybrid bcl-2/immunoglobulin heavy chain transcripts are produced that consist of the 5' half of the bcl-2 mRNA fused to a "decapitated" immunoglobulin heavy chain mRNA. Nucleotide sequence analyses confirmed that the hybrid transcripts continue to encode a normal bcl-2 protein. Our results suggest that t(14;18) translocations alter expression of the bcl-2 gene both by transcriptional activation and by abnormal posttranscriptional regulation of bcl-2 mRNA.
Abelson virus has been used to transform cells of a murine, factor-dependent myeloid cell line (FD). Factor-independent (FI) cell lines were derived, which expressed the viral genome and were tumorigenic in syngeneic mice. Karyotypic analysis of FI cells before and after passage in vivo indicated that the tumorigenic cells were derived from FD cells. Northern gel analysis of mRNA, bioassay of culture supernatants, and the density-independent growth of the FI cells indicated that the transformation had not induced the synthesis of the hemopoietic growth factors normally required to support the FD cells, that is, granulocyte-macrophage CSF or Multi-CSF. The FD and FI cells displayed similar numbers of cell surface receptors for Multi-CSF (IL-3) and GM-CSF. We conclude that Abelson virus transformation of this line from factor-dependence to factor-independence and tumorigenicity did not involve autocrine stimulation.
Retrovirus vectors [direct orientation (DO) vectors] that permit the simultaneous expression of an inserted protein-coding sequence and a dominant-acting selectable marker have been constructed. In these vectors, an internal simian virus 40 or human metallothionein promoter sequence serves to drive the expression of the bacterial neomycin phosphotransferase or guanine-xanthine phosphoribosyltransferase genes, whereas the viral long terminal repeat sequences are utilized to promote expression of inserted sequences. In some of the vectors, the viral 5' splice site, normally used in the biogenesis of the subgenomic env-encoding mRNA, has been eliminated. These vectors yield high transient and stable titers of virus after transfection of viral packaging cell lines, show little or no depression of virus titer with a variety of inserts, and faithfully transmit recombinant proviral sequences to recipient cells. To characterize the expression potential of these vectors, a variety of inserts encoding the alpha and beta subunits of the human major histocompatibility complex class II antigen HLA-DR have been introduced into these vectors. NIH 3T3 cells infected by viruses containing HLA-DR alpha or beta cDNAs express these proteins as shown by immunoprecipitation of metabolically labeled extracts. In addition, through the sequential infection of cells with retrovirus constructions expressing two different selectable markers, both subunits of the class II antigen have been introduced into NIH 3T3 cells. Such infected cells express HLA-DR molecules at the cell surface.
Transgenic mice bearing a cellular myc oncogene coupled to the immunoglobulin heavy-chain enhancer (E mu) exhibit perturbed
B-lymphocyte development and succumb to B lymphoid tumors. To investigate how the enhancer has affected myc expression, we
analyzed the structure and abundance of myc transcripts in tissues of prelymphomatous mice and in the lymphomas. Expression
of the E mu-myc transgene appeared to be confined largely to B lymphoid cells, being dominant in bone marrow, spleen, and
lymph nodes, with no detectable expression in T cells or other hematopoietic lineages examined. The myc transcripts initiated
very predominantly at the normal myc promoters, although use of the more upstream myc promoter was accentuated and an enhancer-associated
promoter may be used infrequently. The level of E mu-myc transcripts in the preneoplastic lymphoid tissues and in the E mu-myc
tumors was not markedly higher than myc RNA levels in proliferating normal lymphocytes. Thus, enforced expression of structurally
normal myc transcripts at only a modestly elevated level has profound biological consequences. The absence of detectable endogenous
c-myc RNA in any tumor, or in preneoplastic bone marrow, supports a negative feedback model for normal c-myc regulation.
Transgenic mice bearing a c-myc oncogene subjugated to the lymphoid-specific immunoglobulin heavy chain enhancer (E mu) develop clonal B lymphoid malignancies, but most young E mu-myc mice lack malignant clones. Their prelymphomatous state has allowed us to examine how constitutive c-myc expression influences B cell development. We find that early stages are overrepresented, even before birth. Pre-B cells of polyclonal origin increase greatly, while B cells develop in reduced number. Both the pre-B and the B cells appear to be in an active state, since they are larger than normal and a greater fraction are in the cell cycle. Enforced myc expression has thus favored proliferation over maturation. Hence, a normal function of c-myc may be to regulate differentiation as well as to promote cell cycling.
Interleukin 3 (IL-3) promotes the survival, proliferation and development of progenitor cells from several distinct haemopoietic lineages and can also stimulate the self-renewal of stem cells. We have explored the mode of action of this growth factor in promoting survival and proliferation, using a multipotent haemopoietic stem cell line FDC-Mix 1. In the absence of IL-3 these cells died within 16-48 h. However, this requirement for IL-3 could be replaced by 12-O-tetradecanoylphorbol-13-acetate (TPA) plus Ca2+ ionophore, which promoted not only survival but also DNA synthesis with no concomitant loss of the multipotential nature of these cells. TPA and Ca2+ ionophore, respectively, could also interact synergistically with IL-3 to promote DNA synthesis. Both IL-3 and TPA stimulated the translocation of protein kinase C (PK-C) from the cytosol to a membrane-bound form in FDC-Mix 1 cells. Previously we suggested that IL-3 can activate the primary metabolism of IL-3-dependent cells so that increased glucose transport and glycolysis lead to maintenance of ATP levels and cellular survival. To investigate whether TPA and, or, Ca2+ ionophore could also influence cellular survival via an activation of glucose uptake we assessed the effects of these agents on hexose transport. TPA +/- Ca2+ ionophore activated hexose transport to the same degree as does IL-3 but these agents cannot superstimulate FDC-Mix 1 hexose transport in cells that already exhibit an activated transport system from preincubation with IL-3. We conclude that IL-3 maintains FDC-Mix 1 cells via its ability to activate PK-C and increase cytosolic levels of Ca2+, and that an IL-3-mediated activation of PK-C may promote cellular survival via its ability to enhance hexose uptake by phosphorylating the glucose transport protein.
Bone marrow stromal cell lines have been isolated that directly support B lymphopoiesis in vitro. Single B-lineage precursors proliferate and differentiate on certain of these stromal cell lines to establish long-term B-lineage cultures. These lymphopoietic stromal cells produce novel soluble factors that support proliferation of in vitro established pre-B cell populations. Lymphoid populations established on lymphopoietic stromal cell lines lack surface Ig-bearing cells, but give rise to surface Ig+ cells when transferred to mixed bone marrow feeder layers. Several stromal lines expressed a B-lineage neoplasia marker detected by the monoclonal antibody MAb6C3. Remarkably, only the 6C3Aghi stromal lines supported long-term proliferation of B-lineage cells. We propose that the 6C3 antigen-bearing molecule may play a role in stromal cell-dependent, pre-B cell proliferation, as well as in neoplastic proliferation of pre-B leukemias.
A retrovirus encoding polyoma middle T antigen has been used to infect a murine hemopoietic cell line (FDC-P1) dependent on either granulocyte-macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (Multi-CSF). A number of cell lines have been established on the basis of their initial ability to proliferate in the absence of added colony-stimulating factor (CSF). The transformed lines display one of three patterns of growth in vitro: those able to grow fully autonomously; those whose proliferation depends on cell density; and those displaying dependence on added CSF regardless cell density. This latter class of cells are reminiscent of the majority of primary myeloid leukemic cells. Unlike parental FDC-P1 cells, all three classes of transformed cells are leukemogenic in syngeneic mice; moreover, they produce variable amounts of GM-CSF which we believe underlies their neoplastic behavior.
Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.
Transgenic mice bearing the cellular myc oncogene coupled to the immunoglobulin mu or kappa enhancer frequently develop a fatal lymphoma within a few months of birth. Since the tumours represent represent both immature and mature B lymphocytes, constitutive c-myc expression appears to be highly leukaemogenic at several stages of B-cell maturation. These myc mice should aid study of lymphoma development, B-cell ontogeny and immunoglobulin regulation.
Specific chromosomal translocations found in distinct neoplasms suggest that genes that flank such breakpoints play a critical role in transformation. We have characterized the t(14;18)(q32;q21) chromosomal translocation present in over 60% of human follicular lymphomas. We exploited an unexpected rearrangement of an Ig heavy-chain gene to clone the chromosomal breakpoint. An element isolated from 18q21 mediated translocations in all four t(14;18) bearing cell lines and in six of 11 follicular lymphomas, but did not normally rearrange in other B or non-B cells. The breakpoints clustered within a small 4.3 kb region on chromosome 18. The breakpoints on chromosome 14 were focused within or immediately 5' to JH. These breakpoints retained the Ig enhancer region close to a new transcriptional unit identified on chromosome segment 18q21. Since none of the cellular oncogenes are known to map to 18q21, cloning this element provides an opportunity to characterize a potentially new transforming gene.
From an acute B-cell leukemia cell line, a DNA probe was obtained that was specific for chromosome 18 and flanked the heavy
chain joining region of the immunoglobulin heavy chain locus on chromosome 14. This probe detected rearrangement of the homologous
DNA segment in the leukemic cells and in follicular lymphoma cells with the t(14:18) chromosome translocation but not in other
neoplastic or normal B or T cells. The probe appears to identify bcl-2, a gene locus on chromosome 18 (band q21) that is unrelated
to known oncogenes and may be important in the pathogenesis of B-cell neoplasms with this translocation.
A new procedure is reported for the staining of DNA, for flow microfluorimetry. It allows the production of stained cell nuclei in a single step by incorporating the DNA stain with a solution of the nonionic detergent Triton-X-100. This method has been found to be applicable to all DNA fluorochromes tested (ethidium bromide, propidium iodide, mithramycin, DAPI, Hoechst 33342). DNA histograms obtained in this way are comparable to those using conventional staining techniques, e.g., ethanol fixation followed by staining. Using this procedure the DNA content distribution of solid tissue or cells from suspension or monolayer cultures can be generated in less than 5 min.
The population dynamics of B lymphocytes and their precursors was followed in normal, adult mice, after killing dividing cells with high doses of hydroxyurea (HU), by immunofluorescence techniques identifying mature B cells (sIgM+), lymphoid cells that show perinuclear staining with rhodamine-conjugated anti-μ antibodies (cIgM+), and Ig-negative precursors the express membrane structures cross-reactive with 'public' antibody idiotypes (Id+, IgM-). In addition, functional reactivities were analyzed by determining in limiting dilution the frequency of mitogen-reactive B cells competent to be activated to clonal proliferation and IgM secretion by 2 different mitogens. HU treatment results in a sharp decrease in the total number of B cells in all organs, and it depletes, in an average of 5 experiments, 80 to 90% of the starting population by 24 to 48 hr in bone marrow (BM), 60 to 80% by 24 to 48 hr in the thoracic duct (TD), and 50 to 60% by 48 to 72 hr in the spleen (SP). Among all BM cells with markers of the B lymphocyte lineage, the greatest depletion was observed in the Id+, IgM- set, and the least affected were cIgM+ cells. Both these and mature sIgM+ cells, although reduced in absolute numbers, actually showed increased frequencies in BM after treatment, whereas the frequencies of sIgM+ cells dropped in TD and remained fairly constant in SP. Lipopolysaccharide- (LPS) and lipoprotein- (LP) reactive B cells were also drastically reduced, to a range of 20 to 30% of control numbers by 48 to 72 hr in BM and TD, and to 50 to 60% by day 4 or 5 in SP. The relative frequencies of mitogen-reactive cells, however, were unchanged or slightly enhanced in HU-treated animals, suggesting that this B lymphocyte population has an average lifetime that is similar to or only slightly longer than that of the majority to B cells present in these organs. These results demonstrate that the majority of peripheral B lymphocytes or their immediate precursors in normal mice have recently divided. Since, on the other hand, not more than 1% of such B cells are large (cycling) blasts, this conclusion suggests that the large numbers of B cells continuously generated in BM are in fact exported to the periphery.
We develop a murine retrovirus shuttle vector system for the efficient introduction of selectable and nonselectable DNA sequences into mammalian cells and recovery of the inserted sequences as molecular clones. Three protocols allow rapid recovery of vector DNA sequences from mammalian cells. Two of the methods rely on SV40 T-antigen-mediated replication of the vector sequences and yield thousands of bacterial transformants per 5 X 10(6) mammalian cells. The majority of plasmids recovered by all three protocols exhibited the proper structure and were as active as the parental vector in the generation of transmissible retrovirus genomes upon transfection of mammalian cells. One of the rescue methods, which relies on "onion skin" replication and excision of an integrated provirus from the host chromosome, enables facile recovery of the chromosomal site of proviral integration. The system was also used to generate, and then efficiently recover, a cDNA version of a genomic insert from the adenovirus E1A region.
A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-psi-, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5' splice site and the AUG that initiates the coding sequence for Pr65gag. Although the parent wild-type proviral clone, pMOV-psi+, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-psi- into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-psi- -transfected cells produce infectious virus. Thus pMOV-psi- has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-psi- stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-psi- appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-psi- can be used to produce helper-free stocks of natural or synthetic defective retroviruses.
Growth and development of haematopoietic cells in vitro require the presence of specific regulatory molecules. Some of these molecular species appear to have a broad specificity, being able to promote the proliferation and differentiation of multipotential cells, as well as megakaryocytic, erythroid and granulocytic-progenitor cells. Such factors are present in medium conditioned by the growth of lectin-stimulated mouse spleen cells or WEHI-3 myelomonocytic leukaemia cells (WEHI-CM). Using WEHI-CM, we and other have been able to obtain permanently growing, non-leukaemic cell lines of a granulocytic or mast cell nature. Significantly, we have found that the factor in WEHI-CM necessary for the growth of these cells has co-purified with the multi-lineage stimulating activity present in WEHI-CM, suggesting that one molecule may be concerned in the development of multiple cell types. We have now used these cells to investigate the mode of action of this haematopoietic cell growth factor and have found that the requirement of this factor for survival and growth may lie in its ability to modulate ATP levels within the cells.