Content uploaded by Celine Mirjolet
Author content
All content in this area was uploaded by Celine Mirjolet on Jun 12, 2018
Content may be subject to copyright.
A preview of the PDF is not available
Oncoprotein expression of E6 and E7 does not prevent 5-fluorouracil (5FU) mediated G1/S arrest and apoptosis in 5FU resistant carcinoma cell lines
Abstract
5-Fluorouracil (5FU) exposure can lead to both G1/S arrest and apoptosis induction which are dependent of P53 induction. The human papilloma virus oncoproteins (HPV), E6 and E7, inactivate respectively P53 and Rb. P53 degradation by E6 protein, leads to lack of G1/S arrest after genotoxic stress. Overexpression of E7 protein prevents P53-induced G1/S arrest following DNA damage. However, few studies have described 5FU effect and efficacy on cancer cell lines presenting HPV 18 positive status. KB cell line and KB3 subline presented wild-type P53 status and difference in 5FU sensitivity. During 5FU exposure, P53 gene and protein expression was increased in both cell lines. E6 and E7 mRNA and protein expression was decreased in KB and KB3. P53 and E6 protein expressions were inversely correlated. 5FU exposure, induced a G1/S arrest which can be maintained or intensified by P53 via P21 induction expression. 5FU exposure has led to apoptosis induction related to P53 induction. In the present study, 5FU exposure was shown to induce G1/S arrest and apoptosis by P53-dependent molecular pathway, in HPV 18 positive cells.
... Nevertheless, neither E6 nor E7 proteins regulated by HPV-18 could induce apoptosis evasion from 5fluorouracil in the head and neck KB carcinoma cells. [76]. Further researches are warranted to fully understand the underlying mechanism by which HPV-16 promotes drug-resistance in non-cancerous infected cells (Fig. 2). ...
Chemoresistance is often referred to as a major leading reason for cancer therapy failure, causing cancer relapse and further metastasis. As a result, an urgent need has been raised to reach a full comprehension of chemoresistance-associated molecular pathways, thereby designing new therapy methods. Many of metastatic tumor masses are found to be related with a viral cause. Although combined therapy is perceived as the model role therapy in such cases, chemoresistant features, which is more common in viral carcinogenesis, often get into way of this kind of therapy, minimizing the chance of survival. Some investigations indicate that the infecting virus dominates other leading factors, i.e., genetic alternations and tumor microenvironment, in development of cancer cell chemoresistance. Herein, we have gathered the available evidence on the mechanisms under which oncogenic viruses cause drug-resistance in chemotherapy.
... Acquired drug resistance is the main retard to the successful treatment of cancer, so it is very important to determine the molecular mechanism related to drug resistance [33]. Several resistance mechanisms to 5FU have been described, including changes related to transport, metabolism, cell cycle and protection of apoptosis [34][35][36][37]. Therefore, further prospecting for overcoming the resistance of chemotherapy drugs to cancer cells will be used in combination with compound drugs to increase the effectiveness of cancer treatment. ...
Coronarin D (CD) is one of the main components of Hedychium coronarium rhizome, which has therapeutic potential by reducing cell proliferation in cancer cells. However, the mechanism of CD to 5-fluorouracil (5FU) oral cancer cell remain unclearly. This study discusses the CD to 5FU chemoresistance oral squamous cell carcinoma (OSCC) biochemical mechanisms and possibly pathways to inhibit multiplication in oral cancer. The effect of CD-treated 5FU-chemoresistance human oral cancer cell lines were subjected to MTT assay, cell cycle assay, DAPI assay, annexin-V/PI double staining assay and mitochondrial membrane potential measurement. Furthermore, western blotting was performed to assess the effect of CD on the expression levels of apoptosis related protein and MAPK signaling pathway. The results of the study evidenced that CD reduced viability of 5FU cancer cells in a dose- and time-dependent manner compared with control. The cytotoxic effect of CD lead to cell cycle arrest in the G2/M phase and induced apoptosis in both internal and external pathways. CD induces apoptosis by enhancing phosphorylation of JNK, further exploring the combination of CD and SP600125 reduced the overexpression of phosphate JNK levels. The mechanism of action of CD in 5FU on human oral cancer cells is reported for the first time and can hopeful to be a potential therapeutic agent for 5FU against human oral cancer cells.
... DNA-damaging reagents such as 5-FU are the most commonly used chemotherapy drugs for clinical cancer therapy, as they induce cell cycle arrest to prevent cell proliferation and trigger cell apoptosis in cancer cells 15 . The therapeutic effect of chemotherapy drugs is highly dependent on the status of TP53 in cancer cells, which is concerning as p53 pathway mutations occur frequently in human cancer 16,17 . ...
Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. 5-Fluorouracil (5-FU) is widely used in the treatment of cancers, but its antineoplastic activity is limited in drug-resistant cancer cells. To investigate the detailed mechanism of 5-FU resistance, we developed a model of 5-FU-resistant cells from HCT-8 cells, a well-established colorectal cancer cell line. We found that the drug-resistant cells demonstrated high expression of TCF4 and β-catenin, indicating an upregulated Wnt pathway. A microarray analysis revealed that the suppression of the checkpoint kinase 1 (CHK1) pathway explained the resistance to 5-FU, especially in p53 wild-type cancer cells such as HCT-8. Our data also demonstrated that the CHK1 pathway is suppressed by the Wnt pathway in 5-FU-resistant cells. In summary, we have discovered a novel mechanism for 5-FU resistance mediated by histone deacetylation, which also revealed the crosstalk between the Wnt pathway and CHK1 pathway.
... Similarly, cisplatin administration has been shown to significantly increase levels of MDA [9,10] and reduce the number of hippocampal neurons, changes which are concomitant with memory deficits [10]. Furthermore, previous studies have reported that 5-FU can induce expression of p21, a marker of cell damage, in cell culture [54]. Our study revealed that 5-FU per se significantly enhanced p21 positive cell numbers when compared to the controls. ...
5-fluorouracil or 5-FU (a chemotherapeutic medication) has been revealed to induce memory deficits in many cancer patients. Asiatic acid (AA) is a triterpenoid extract from Centella asiatica. This compound can ameliorate intracellular oxidative stress caused by chemotherapy drugs. Recent studies have shown that AA is capable of inhibiting neuronal generation and memory deficit produced by 5-FU chemotherapy. This study aimed to assess the molecular mechanisms of AA related to hippocampal neurogenesis and memory in rats receiving 5-FU. Male Sprague Dawley rats were given AA (30 mg/kg) orally and given 5-FU (25 mg/kg) by i.v. injection 5 times. Some rats were given AA for 20 days before and during 15-FU treatment (preventive), some received AA for 20 days after 5-FU treatment (recovery), and some underwent treatment with AA throughout the time of the experiment (throughout) for 40 days. Treatment with 5-FU caused significant reductions in Notch1, sex determining region Y-box 2 (SOX2), nestin, doublecortin (DCX), and nuclear factor erythroid 2-related factor 2 (Nrf2) levels within the hippocampus. In addition, 5-FU significantly increased p21 positive cell number in the subgranular zone (SGZ) and malondialdehyde (MDA) levels in the hippocampus. Administration with both AA and 5-FU in prevention and throughout was able to prevent decreases in Notch1 SOX2, nestin, DCX, and Nrf2 caused by 5-FU. Treatment with AA also led to decreases in p21 positive cells and MDA levels in the hippocampus. These findings exhibit that AA has the ability to counteract the down-regulation of neurogenesis within the hippocampus and memory deficits caused by 5-FU via inhibiting oxidative stress and increasing neuroprotective properties.
... Up-regulation of cellular inhibitor of apoptosis protein 2 (c-IAP2) in human oral keratinocytes 20 , as well as AP-1 proteins in cervical carcinoma cell lines 21 , has been associated with the expression of the E6 oncogene. However, the expression of HPV18 E6 and E7 in the head and neck KB carcinoma cell line could not prevent apoptosis induced by 5fluoruracil 22 33 (2007) had acquired a few copies of HPV16 by natural infection 23 , to address this issue. A multi-drug resistant SiHa cell line (SiHaR) selected from SiHa parental cells 24 was compared to its parental line on patterns of antiapoptotic gene expression and on HPV oncogene expression. ...
Alteration of the apoptosis pathway, as well as the presence of human papilloma virus (HPV), has been linked to the proliferative capacity and drug resistant phenotype of SiHa cervical cancer. We investigated the roles of E6 and E7 HPV oncoproteins in the expression of apoptosis regulating genes in cervical cancer cells that contain the characteristics of apoptosis resistance, and also their correlation with resistance to various apoptosis inducing agents. The expression of the sets of apoptosis regulating genes in both extrinsic (receptor) and intrinsic (non-receptor) pathways were monitored in parental SiHa and multi-drug resistant SiHa (SiHaR) cell lines by RNase protection assay and RT-PCR. An increase in gene expression of intrinsic pathway anti-apoptotic protein Bcl-XL was seen, both at the mRNA and protein levels, in SiHaR compared with SiHa cells, whereas the expression of the genes involved in the extrinsic pathway remained unchanged. SiHaR cells also expressed higher levels of E6 and E7 than did SiHa. Caspase 3 activity was lower in SiHaR compared with that in SiHa cells. A colony formation assay demonstrated enhanced resistance of SiHaR cells to several types of apoptosis inducing agents, including etoposide, doxorubicin, cisplatin, and γ-radiation. Transfection of HPV-negative C33a cells with HPV oncogenes, E7 in particular, induced transcription of Bcl-XL, supporting the role of HPV oncoproteins in affording chemo-radio resistance in cervical cancer.
... The tumor suppressor protein p53, a transcription factor controls the cell cycle (and arrests it in case of DNA damage). Inhibition of tumor growth through cell cycle arrest and induction of apoptosis are functionally related to p53 (Kobayashi et al., 2002; Didelot et al., 2003). Luteolin could mediate p53 stabilization and accumulation , which induces apoptosis and prevents cell proliferation in many cancer cell lines, including breast cancer (Momtazi-Borojeni et al., 2013), Eca109 (Wang et al., 2012b), gastric cancer AGS (Wu et al., 2008), HT-29 colon cancer (Lim do et al., 2007), and head and neck and lung cancer (Amin et al., 2010). ...
Many food-derived phytochemical compounds and their derivatives represent a cornucopia of new anticancer compounds. Despite extensive study of luteolin, the literature has no information on the exact mechanisms or molecular targets through which it deters cancer progression. This review discusses existing data on luteolin's anticancer activities and then offers possible explanations for and molecular targets of its cancer-preventive action. Luteolin prevents tumor development largely by inactivating several signals and transcription pathways essential for cancer cells. This review also offers insights into the molecular mechanisms and targets through which luteolin either prevents cancer or mediates cancer cell death.
... Whether this effect was associated with an accumulation of viral oncogene E6/E7 and WNT activation or lower vulnerability of HPV-16-associated HNSCC needs to be evaluated. On the other hand, it was demonstrated that oncoprotein expression of E6 and E7 did not prevent 5-FU mediated G 1 /S arrest and apoptosis in 5-FU-resistant carcinoma cell lines (50). Liu and collegues found that 5-FU presented some inhibitory effects on the E6 and E7 oncoproteins of HPV16 in laryngeal cancer cells. ...
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. In several tumour entities, the tyrosine kinase receptor c-KIT is associated with tumour transformation in the epithelial tissue in cases of aberrant expression. Furthermore, tumour development and dissemination are a result of dysregulated cellular pathways such as the WNT/β-catenin pathway. β-Catenin is a multifunctional protein within the canonical WNT signalling pathway and a pivotal factor for the stabilization of cell-cell interactions. In malignant tissues, β-catenin triggers tumour proliferation and progression. The aim of this study is to investigate the expression patterns of c-KIT and β-catenin in human papillomavirus-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutical agents docetaxel and 5-fluorouracil (5-FU).
We incubated the tumour cell lines with docetaxel (5 μmol/ml) and 5-FU (1 μmol/ml) and detected β-catenin and c-KIT by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h.
We found a reliable trend towards decreased β-catenin expression levels in p16-positive and p16-negative tumour cell lines when incubated with docetaxel, in addition to induced apoptotic effect. At best, 5-FU had a slight influence on the alteration of the expression of β-catenin. Dose escalation of docetaxel and 5-FU had no statistically significant effect on the expression of β-catenin or c-KIT. In HPV-negative HNSCC, a reduced expression level of β-catenin and c-KIT was detected in an incubation period-dependent manner. p16-transformed SCC (CERV196) cells were characterized by a reduced susceptibility to docetaxel induced alteration of β-catenin expression.
We were unable to confirm the clinically-substantiated increased chemosensitivity of p16-positive tumour cells in vitro. Extended studies and clinical trials are needed to investigate these findings further in HPV-associated HNSCC.
... The delayed and continuing effects of chemotherapy treatment found in the present study may provide an explanation for the patient reports of prolonged cognitive impairment. The mechanism behind these effects remains unclear but it has previously been reported that 5-FU exposure induces cell cycle arrest at the G1/S transition and apoptosis by a P53-dependent molecular pathway, in HPV 18 positive cells [12]. Similarly the chemotherapy agents BCNU and Cisplatin have been found to have longer term effects on cell survival [13,48]. ...
As one of the frequent malignancies, breast cancer (BCa) is the foremost reason for cancer-related deaths among women. The role of Human papillomavirus (HPV) in chemoresistance has rarely been investigated in previous studies. The current study sets out to the possible role of HPV in BCa chemoresistance. In this research, 90 BCa tissue and 33 normal breast tissue were collected. We evaluated the presence of the HPV genome along with the viral (E2, E6, E7) and cellular gene expression associated with cell resistance to death. Statically significant differences in the prevalence of HPV between the BCa group (25.2% or 23/90) and the control group (21.8% or 7/32) were not found. HPV-16 and HPV-18 genotypes were the abundant HPV genotypes. Resistance to the Adriamycin-Cyclophosphamide (AC), paclitaxel regimen was elevated in the HPV- group (56/70) in comparison to the HPV+ group (14/70). Nevertheless, there was no significant difference in the prevalence of resistance to AC + paclitaxel + triple-negative breast cancer combination therapy between the HPV+ group (9/20) and in the HPV- group (11/20). In the BCa group in contrast to the control group, the expression level of Bcl-2, BCL-XL, and c-IAP2 demonstrated a significant decrease, while, the expression level of cytochrome C and caspase 3 was significantly increased. This study suggests that HPV infection might contribute to BCa chemoresistance through disrupt cellular genes involved in cell death.
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the leading cause of coronavirus disease-2019 (COVID-19), is an emerging global health crisis. Lung cancer patients are at a higher risk of COVID-19 infection. With the increasing number of non-small-cell lung cancer (NSCLC) patients with COVID-19, there is an urgent need of efficacious drugs for the treatment of COVID-19/NSCLC.
Methods: Based on a comprehensive bioinformatic and systemic biological analysis, this study investigated COVID-19/NSCLC interactional hub genes, detected common pathways and molecular biomarkers, and predicted potential agents for COVID-19 and NSCLC.
Results: A total of 122 COVID-19/NSCLC interactional genes and 21 interactional hub genes were identified. The enrichment analysis indicated that COVID-19 and NSCLC shared common signaling pathways, including cell cycle, viral carcinogenesis, and p53 signaling pathway. In total, 10 important transcription factors (TFs) and 44 microRNAs (miRNAs) participated in regulations of 21 interactional hub genes. In addition, 23 potential candidates were predicted for the treatment of COVID-19 and NSCLC.
Conclusion: This study increased our understanding of pathophysiology and screened potential drugs for COVID-19 and NSCLC.
The adenovirus E1A gene product, the simian virus 40 large tumor antigen, and the human papillomavirus E7 protein share a short amino acid sequence that constitutes a domain required for the transforming activity of these proteins. These sequences are also required for these proteins to bind to the retinoblastoma gene product (pRb). Recent experiments have shown that E1A can dissociate complexes containing the transcription factor E2F bound to pRb, dependent on this conserved sequence element. We now show that the E7 protein and the simian virus 40 large tumor antigen can dissociate the E2F-pRb complex, dependent on this conserved sequence element. We also find that the E2F-pRb complex is absent in various human cervical carcinoma cell lines that either express the E7 protein or harbor an RB1 mutation, suggesting that the loss of the E2F-pRb interaction may be an important aspect in human cervical carcinogenesis. We suggest that the ability of E1A, the simian virus 40 large tumor antigen, and E7 to dissociate the E2F-pRb complex may be a common activity of these viral proteins that has evolved to stimulate quiescent cells into a proliferating state so that viral replication can proceed efficiently. In circumstances in which a lytic infection does not proceed, the consequence of this action may be to initiate the oncogenic process in a manner analogous to the mutation of the RB1 gene.
The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)
The E6 protein of human papillomavirus types 16 and 18 (HPV-16 and HPV-18) can stably associate with the p53 protein in vitro. In the presence of rabbit reticulocyte lysate, this association leads to the specific degradation of p53 through the ubiquitin-dependent proteolysis system. We have examined the E6-p53 complex in more detail and have found that association of E6 with p53 is mediated by an additional cellular factor. This factor is present in rabbit reticulocyte lysate, primary human keratinocytes and in each of five human cell lines examined. The factor is designated E6-AP, for E6-associated protein, based on the observation that the E6 proteins of HPV-16 and 18 can form a stable complex with the factor in the absence of p53, whereas p53 association with the factor can be detected only in the presence of E6. Gel filtration and coprecipitation experiments indicate that E6-AP is a monomeric protein of approximately 100 kDa.
DNA of a new papillomavirus type was cloned from a cervical carcinoma biopsy. Two EcoRI clones of 7.8 and 6.9 kb in length were obtained, the latter contained a 900-bp deletion. The BamHI fragments of both clones were used to characterize the DNA. It represents a distinct type of papillomavirus as determined by its size, its cross-hybridization with DNA of other papillomavirus types under conditions of low stringency only, the co-linear alignment of its genome with HPV 6 and HPV 16 prototypes and its occasional occurrence as oligomeric episomes. We tentatively propose to designate it as HPV 18. DNA hybridizing with HPV 18 under stringent conditions was detected in 9/36 cervical carcinomas from Africa and Brazil, in 2/13 cervical tumors from Germany and 1/10 penile carcinomas. Benign tumors (17 cervical dysplasias, 29 genital warts), eight carcinomata in situ and 15 biopsies of normal cervical tissue were devoid of detectable HPV 18 DNA. HPV 18-related DNA was found, however, in cells of the HeLa, KB and C4-1 lines all derived from cervical cancer. The state of the viral DNA was investigated in four cervical cancer biopsies. The data reveal that the DNA might be integrated into the host cell genome. One tumor provided evidence for head to tail tandem repeats some of which persisted as circular episomes.
Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.
Hallmarks of HPV infection include a restricted tropism for human epithelial cells and a viral life cycle tightly linked to the differentiation program of the host keratinocyte. This particular viral cycle has hampered the study of the HPV vegetative life cycle for decades, due to the lack of suitable in-vitro culture conditions. The tissue and differentiation dependence seems to be dictated by viral transcription rather than viral DNA replication. Indeed, viral transcription is restricted to epithelial cells of human origin, more specifically to keratinocytes. In contrast, HPV genomes can replicate in various undifferentiated cell lines regardless of their natural permissiveness to infection, as long as the viral replication proteins E1 and E2 are expressed.
The HPVs associated with anogenital cancers encode two oncoproteins, E6 and E7. Both E6 and E7 can form specific complexes with tumour suppressor gene products. The E7 protein binds to the retinoblastoma tumour suppressor gene product pRB, with a preference for the underphosphorylated, "active" form of pRB. The E7 proteins derived from the "high risk" HPVs bind to pRB with a higher affinity than the E7 proteins from the "low risk" HPVs. The "high risk" HPV E6 proteins can associate with the p53 tumour suppressor protein. This interaction promotes the degradation of p53 in vitro, which presumably accounts for the very low levels of p53 in cervical carcinoma cell lines. The functional inactivation of pRB and p53 by the HPV oncoproteins E7 and E6, respectively, are likely to be important steps in cervical carcinogenesis, since mutations in the RB and p53 genes were detected in HPV negative but not HPV positive cervical carcinoma cell lines. Cytogenetic studies strongly suggest, however, that additional chromosomal changes may be necessary for carcinogenic progression of HPV induced anogenital lesions.
Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.
Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.
The human genomic clone pb2m13 contains a functional beta 2-microglobulin (B2m) gene, which upon transfection is readily expressed in murine fibroblasts. Here we report the nucleotide sequence of the human beta 2m gene and of a nearly full length cDNA clone. A comparison with the murine beta 2m gene reveals that exon/intron boundaries are absolutely conserved. In the protein-coding regions the similarity is 70%. As far as intron sequences of the murine beta 2m gene are available, no significant similarity between human and murine genes is observed. The transcriptional start site of the human beta 2m gene was determined by S1 mapping, and comparison with the nearly full length cDNA clone now defines the transcriptional unit of the beta 2m gene. In the 5' region of the gene strong clustering of the usually underrepresented CpG dinucleotide is found resembling a similar overrepresentation in the 5' regions of the major histocompatibility complex class I genes.