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The development and progression of malignant tumors likely result from consecutive accumulation of genetic alterations, including dysfunctional tumor suppressor genes. However, the signaling mechanisms that underlie the development of tumors have not yet been completely elucidated. Discovery of novel tumor-related genes plays a crucial role in our...
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... which was identified as a key regulator of the cell cycle, was shown to be inactivated in various types types of of human human tumors tumors [18]. [18]. Thus, Thus, the the presence presence of of tumor tumor suppressor suppressor genes genes was was suggested suggested by by cell hybrid hybrid studies studies and and LOH LOH analysis analysis [19–25]. [19 – 25]. Microcell-mediated Microcell-mediated chromosome chromosome transfer transfer (MMCT), (MMCT), which which is is used used to to transfer transfer a a chromosome from from normal normal somatic somatic cells cells into into human human tumor tumor cells, cells, is is also also used used as as another another approach approach for for efficient identification identification of of tumor tumor suppressor suppressor genes genes [26,27]. [26,27]. We We established established a direct a direct approach approach for for identification of a of chromosome a chromosome carrying carrying tumor tumor suppressor suppressor gene(s) gene(s) by by introducing introducing individual individual normal normal chromosomes into into tumor tumor cells cells [28–41]. [28 – 41]. In In this this review, review, we we outline outline a general a general strategy strategy for mapping for mapping the localization the and identification and identification of a functional of a functional tumor tumor suppressor suppressor gene gene with with MMCT. MMCT. mapping and identifying tumor suppressor genes. increase utilization of MMCT as a systematic approach for cancer research, we constructed constructed a library library of mouse A9 cells cells containing containing a single human chromosome, allowing allowing any any human human chromosome chromosome to to be be introduced introduced into into target target recipient recipient cells cells such such as as cancer cancer cells. cells. The The strategy strategy we used we used for the for mouse the mouse A9 monochromosomic A9 monochromosomic library library containing containing a single a single human human chromosome chromosome is outlined is outlined in Figure in Figure 1. First, 1. First, we transfected we transfected plasmids plasmids with with pSV2 pSV2 neo , bsr neo , , and bsr , and pGK pGK neo into neo into normal normal human human fibroblast fibroblast cells, cells, resulting resulting in in random random integration integration of of these dominant dominant selectable markers into human human chromosomes. chromosomes. Neomycin- and blasticidin blasticidin S S hydrochloride-resistant hydrochloride-resistant clones clones were were isolated isolated and and fused fused to to mouse mouse A9 A9 cells, cells, producing producing many many human/mouse human/mouse hybrid clones that contain selectable selectable marker-tagged marker-tagged human human chromosomes. chromosomes. Finally, Finally, transfer of dominant dominant selectable selectable marker-tagged human chromosomes obtained from those hybrid cells into mouse A9 cells was performed performed with microcell fusion. To investigate the status of the introduced human chromosome, karyotyping and fluorescent fluorescent in situ hybridization analysis of the isolated A9 microcell hybrid clones were were performed. performed. We We have have now now generated generated A9 A9 hybrids hybrids that each contain a human chromosome, except the the Y Y chromosome chromosome [36,42,43]. ...
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... Moreover, early studies in the 90s utilized MMCT to map tumor growth suppression and anti-metastatic activities on human chromosomes by reverting a chromosomal loss or inducing a specific chromosomal gain in various cancer cell lines (Oshimura et al. 1989;Yamada et al. 1990;Kugoh et al. 1990;Ogata et al. 1993;Tanaka et al. 1998). As a result, a considerable number of human, mouse, and rat cell lines that harbor an extra single human chromosome were generated (reviewed by Kugoh et al. 2016). Functional studies on these cell lines led to the hypothesis that loss of a specific chromosome or chromosome arm could be a strategy to silence tumor-suppressing genes located on that chromosome. ...
... These anti-tumorigenic effects of chr3 gain were subsequently attributed to multiple tumor-suppressing genes on chr3p, including several telomerase repressors (Uzawa et al. 1998;Abe et al. 2010;Nishio et al. 2015). Furthermore, transferring either whole chromosomes or parts of human chromosomes 1-8, 10-13, 16-20, 22, X, or Y into different cancer or non-transformed immortalized cell lines mainly inhibited the proliferative, tumorigenic and/or metastatic potential of the recipient cell line (reviewed in Yoshida et al. 2000;Meaburn et al. 2005;and Kugoh et al. 2016). However, only in a few cases could the tumor-suppressing effects of a specific chromosome transfer be narrowed down to a single gene or gene cluster (Dong et al. 1995;Yoshida et al. 1999;Seraj et al. 2000;Goldberg et al. 2003). ...
... Analyses of transformed and non-transformed human cell lines with a single trisomic chromosome (Supplemental table 1) revealed that a chromosome gain often leads to increased CIN, replication stress, as well as global transcriptomic and proteomic changes (Phillips et al. 2001;Phillips et al. 2001;Nawata et al. 2011;Stingele et al. 2012;Dürrbaum et al. 2014;Nicholson et al. 2015;Passerini et al. 2016). Furthermore, in line with earlier observations (Yoshida et al. 2000;Meaburn et al. 2005;Kugoh et al. 2016), these recent studies also showed that nearly all single chromosomal gains negatively impact cellular transformation and metastasis formation. However, this generally appears not to be due to expression of tumor-suppressing genes on the gained chromosome, but rather due to the stresses associated with the gene expression imbalances (Vasudevan et al. 2020). ...
An abnormal chromosome number, or aneuploidy, underlies developmental disorders and is a common feature of cancer, with different cancer types exhibiting distinct patterns of chromosomal gains and losses. To understand how specific aneuploidies emerge in certain tissues and how they contribute to disease development, various methods have been developed to alter the karyotype of mammalian cells and mice. In this review, we provide an overview of both classic and novel strategies for inducing or selecting specific chromosomal gains and losses in human and murine cell systems. We highlight how these customized aneuploidy models helped expanding our knowledge of the consequences of specific aneuploidies to (cancer) cell physiology.
... MMCT. Chromosome transfer via chromosome engineering was performed as previously described (14). A9(neo9q) or A9(neo4) cells were treated with 0.05 µg/ml colcemid at 37˚C for 48 h to induce formation of micronuclei, which were then purified by cytochalasin B (10 µg/ml) digestion and centrifugation at 11,900 x g for 60 min at 34˚C. ...
... One week after fusion, no viable cells were observed. Two weeks after fusion, rapidly proliferating clones were isolated and maintained by serial passaging, as previously described (14). ...
Bladder cancer is divided into two molecular subtypes, luminal and basal, which form papillary and nodular tumors, respectively, and are identifiable by gene expression profiling. Although loss of heterozygosity (LOH) of the long arm of human chromosome 9 (9q) has been observed in the early development of both types of bladder cancer, the functional significance of LOH remains to be clarified. The present study introduced human chromosome 9q into basal bladder cancer cell line, SCaBER, using microcell-mediated chromosome transfer to investigate the effect of LOH of 9q on molecular bladder cancer subtypes. These cells demonstrated decreased proliferation and migration capacity compared with parental and control cells. Conversely, transfer of human chromosome 4 did not change the cell phenotype. Expression level of peroxisome proliferator-activated receptor (PPAR)γ, a marker of luminal type, increased 3.0-4.4 fold in SCaBER cells altered with 9q compared with parental SCaBER cells. Furthermore, the expression levels of tumor suppressor PTEN, which regulates PPARγ, also increased in 9q-altered cells. These results suggested that human chromosome 9q may carry regulatory genes for PPARγ that are involved in the progression of neoplastic transformation of bladder cancer.
... We have previously reported that PITX1, a negative regulator of hTERT, interacts with zinc finger CCHC-type containing 10 (ZCCHC10) to regulate hTERT transcription, and both genes are encoded together in the 5q31.1 region 44 . Therefore, chromosome engineering technologies that can transfer an entire chromosome or chromosomal fragments, which contain the essential genomic context for regulation of gene transcription, may enable elucidation of tumor suppressor functions by specific chromosomal domains 45 . ...
... Cell culture. Mouse A9 was purchased from the American Type Culture Collection (ATCC) (Manassas, VA), and A9 microcell hybrid cells containing chromosomes were established in our laboratory 20,45 . A9 microcell hybrid cells containing human chromosomes were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 800 µg/mL G418 (Calbiochem, La Jolla, CA, USA). ...
Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene (hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.
... We have previously reported that PITX1, a negative regulator of hTERT, interacts with zinc nger CCHC-type containing 10 (ZCCHC10) to regulate hTERT transcription, and both genes are encoded together in the 5q31.1 region 42 . Therefore, chromosome engineering technologies that can transfer an entire chromosome or chromosomal fragments, which contain the essential genomic context for regulation of gene transcription, may enable elucidation of tumor suppressor functions by speci c chromosomal domains 43 . ...
Frequent loss of heterozygosity (LOH) on the short arm of human chromosome 3 (3p) region has been found in pancreatic cancer (PC), which suggests the likely presence of tumor suppressor genes in this region. However, the functional significance of LOH in this region in the development of PC has not been clearly defined. The human telomerase reverse transcriptase gene ( hTERT) contributes to unlimited proliferative and tumorigenicity of malignant tumors. We previously demonstrated that hTERT expression was suppressed by the introduction of human chromosome 3 in several cancer cell lines. To examine the functional role of putative TERT suppressor genes on chromosome 3 in PC, we introduced an intact human chromosome 3 into the human PK9 and murine LTPA PC cell lines using microcell-mediated chromosome transfer. PK9 microcell hybrids with an introduced human chromosome 3 showed significant morphological changes and rapid growth arrest. Intriguingly, microcell hybrid clones of LTPA cells with an introduced human chromosome 3 (LTPA#3) showed suppression of mTert transcription, cell proliferation, and invasion compared with LTPA#4 cells containing human chromosome 4 and parental LTPA cells. Additionally, the promoter activity of mTert was downregulated in LTPA#3. Furthermore, we confirmed that TERT regulatory gene(s) are present in the 3p21.3 region by transfer of truncated chromosomes at arbitrary regions. These results provide important information on the functional significance of the LOH at 3p for development and progression of PC.
... PITX1 is involved in the early development of the lower limbs,148 and is upregulated in patients with FSHD. 53 PITX1 regulates the expression of the IFN gene family involved in the activation of the innate immune response against viral infection and is a suppressor of both RAS and tumorigenicity.149 Furthermore, PITX1 is also known to activate components of the p53 pathway causing cell cycle arrest and apoptosis,150 and to induce MURF1 and ATROGIN1.22 ...
Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is caused either by the contraction of the D4Z4 macrosatellite repeat at the distal end of chromosome 4q to a size of 1 to 10 repeat units (FSHD1) or by mutations in D4Z4 chromatin modifiers such as Structural Maintenance of Chromosomes Hinge Domain Containing 1 (FSHD2). These two genotypes share a phenotype characterized by progressive and often asymmetric muscle weakening and atrophy, and common epigenetic alterations of the D4Z4 repeat. All together, these epigenetic changes converge the two genetic forms into one disease and explain the derepression of the DUX4 gene, which is otherwise kept epigenetically silent in skeletal muscle. DUX4 is consistently transcriptionally upregulated in FSHD1 and FSHD2 skeletal muscle cells where it is believed to exercise a toxic effect. Here we provide a review of the recent literature describing the progress in understanding the complex genetic and epigenetic architecture of FSHD, with a focus on one of the consequences that these epigenetic changes inflict, the DUX4‐induced immune deregulation cascade. Moreover, we review the latest therapeutic strategies, with particular attention to the potential of epigenetic correction of the FSHD locus.
... Although it is known that expression of hTERT is regulated by various activating and repressing transcription factors and epigenetic modification [13,14], the underlying molecular mechanisms that are involved in regulation of hTERT transcription during cellular differentiation and cancer development remains unclear. We previously confirmed that human chromosomes 3, 5, and 10 carry hTERT regulatory genes using microcell mediated chromosome transfer (MMCT) [15]. In particular, we identified paired-like homeodomain 1 (PITX1) as a novel hTERT suppressor gene, located on human chromosome 5 by a combination of MMCT and gene expression profile analysis. ...
... RNA isolation and reverse transcriptase (RT)-PCR was performed as described previously [15]. mRNA expression of PITX1 hTERT, ZCCHC10 was analyzed using specific primers: PITX1: forward; 5'-GCTACCCCGACATGAGCA, reverse; 5'-GTTACGCTCGCGCTTACG-3', hTERT: forward; 5'-GCCTTCAAGAGCCACGTC-3', reverse; 5'-CCACGAACTGTCG CATGT-3', ZCCHC10: forward; 5'-TGGACTTATGAATGCACAGGAA-3', reverse; 5'-CT ACATTGGTTTCTCCAATGCTT-3'. p21: forward; 5'-TGGAGACTCTCAGGGTCGAAA, reverse; 5'-GGCGTTTGGAGTGGTAGAAATC-3'. ...
Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, suggesting that this enzyme activity contributes to an unlimited replication potential of cancer cells via regulation of telomere length. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is the functional unit that suppresses hTERT transcription, and may play a crucial role as a novel tumor suppressor complex.
... We previously confirmed that human chromosomes 3, 5, and 10 carry hTERT regulatory genes using microcell mediated chromosome transfer (MMCT) [11]. In particular, we identified paired-like homeodomain 1 (PITX1) as a novel hTERT suppressor gene, located on human chromosome 5 by a combination of MMCT and gene expression profile analysis. ...
Telomerase is a ribonucleoprotein ribonucleic enzyme that is essential for cellular immortalization via elongation of telomere repeat sequences at the end of chromosomes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase holoenzyme, is a key regulator of telomerase activity. Telomerase activity, which has been detected in the majority of cancer cells, is accompanied by hTERT expression, resulting in continuous cell division that is a crucial factor for malignant transformation. Thus, hTERT is an attractive target for cancer-specific treatments. We previously reported that pared-like homeodomain 1 (PITX1) is a negative regulator of hTERT through direct binding to the hTERT promoter. However, the mechanism by which the function of PITX1 contributes to transcriptional silencing of the hTERT gene remains to be clarified. Here, we show that PITX1 and zinc finger CCHC-type containing 10 (ZCCHC10) proteins cooperate to facilitate the transcriptional regulation of the hTERT gene by functional studies via FLAG pull-down assay. Co-expression of PITX1 and ZCCHC10 resulted in inhibition of hTERT transcription, in melanoma cell lines, whereas mutate-deletion of homeodomain in PITX1 that interact with ZCCHC10 did not induce similar phenotypes. In addition, ZCCHC10 expression levels showed marked decrease in the majority of melanoma cell lines and tissues. Taken together, these results suggest that ZCCHC10-PITX1 complex is a new functional unit that suppresses hTERT transcription, and plays a crucial role in cancer development through regulation of telomerase activity.
... The notion of a chromosomal role in cancer development was postulated by Theodore Boveri over 100 years ago (41). Current progress in molecular genetics and biotechnology such as fluorescent in situ hybridization, whole-genome profiling, comparative chromosomal mapping and synthetic chromosome approach, have allowed us a better understanding of the role of chromosomes in cancer evolution (41)(42)(43)(44). The arrangement of genetic materials in eukaryotic cells is significantly different from that of prokaryotic organisms in that it reflects the evolution-dependent higher stability of eukaryotic chromosomes and more complex regulation of gene activity (45). ...
Our review compares evolution of cancer in the human body to the origin of new species from a common ancestor organism with respect to the theory of Charles Darwin. Moreover, the functional role of the tumor microenvironment as a selective pressure actively participating in cancer progression is also demonstrated. Evolutionary aspects of tumor growth and invasion from the point of view of modern therapeutic challenges and opportunities in precision personalized medicine are also discussed. Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
... However, this investment in LOH mapping failed to produce the expected avalanche of dominant driver genes, and with the exception of a few genes such as TP53, mutation rates in known drivers did not correlate with the percentage of LOH at the locus (Ryland et al., 2015). Complementary functional approaches such as microcell-mediated chromosome transfer also did not identify single genes with frequent biallelic inactivation events, although many candidates did demonstrate the capacity to functionally compensate regions of gene deletion (Kugoh et al., 2015) ...
Loss of heterozygosity (LOH) is a genetic event frequently observed in many cancer types. The loss of one allele of a genetic locus can have multiple possible functional effects including haploinsufficiency, loss of gene expression and being the second ‘hit’ that unmasks a recessive tumour suppressor gene. LOH can be caused by mitotic errors, chromothripsis, gene conversion and inappropriate repair of DNA (deoxyribonucleic acid) breaks. The methods for detecting LOH are evolving from single locus assays such as microsatellite analysis, to accurate and sensitive genome‐wide assays including single‐nucleotide polymorphism arrays and massively parallel DNA sequencing. LOH is an important tool to aid in the discovery of novel tumour suppressor genes and now is gaining importance as a biomarker for clinical decision making in certain contexts.
Key Concepts
Loss of heterozygosity is a common genetic event in cancer whereby one allele is lost, leading to part of the genome appearing homozygous in the tumour where heterozygous in matching normal DNA.
Allelic imbalance is different from LOH: both alleles are still present but are in different numbers of copies.
Regions of LOH can be copy number neutral or show copy number loss.
Whole‐chromosome LOH can be caused by mitotic nondisjunction.
Defects in homologous recombination repair lead to increased levels of LOH in cancer.
The two‐hit hypothesis describes the inactivation of both copies of a recessive tumour suppressor gene in cancer; LOH can be one such hit.
Haplo‐insufficiency is where a gene cannot perform its function normally when present as only one copy and is a likely reason for selection of some LOH events.
Telomerase is a ribonucleoprotein ribonucleic enzyme that elongates telomere repeat sequences at the ends of chromosomes and contributes to cellular immortalization. The catalytic component of telomerase, human telomerase reverse transcriptase (hTERT), has been observed to be reactivated in immortalized cells. Notably, most cancer cells have been found to have active hTERT mRNA transcription, resulting in continuous cell division, which is crucial for malignant transformation. Therefore, discovering mechanisms underlying the regulation of hTERT transcription is an attractive target for cancer-specific treatments.
Loss of heterozygosity (LOH) of chromosome 3p21.3 has been frequently observed in human oral squamous cell carcinoma (OSCC). Moreover, we previously reported that HSC3 OSCC microcell hybrid clones with an introduced human chromosome 3 (HSC3#3) showed inhibition of hTERT transcription compared with the parental HSC3 cells. This study examined whether hTERT transcription regulators are present in the 3p21.3 region. We constructed a human artificial chromosome (HAC) vector (3p21.3-HAC) with only the 3p21.3-p22.2 region and performed functional analysis using the 3p21.3-HAC. HSC3 microcell hybrid clones with an introduced 3p21.3-HAC exhibited significant suppression of hTERT transcription, similar to the microcell hybrid clones with an intact chromosome 3. In contrast, HSC3 clones with truncated chromosome 3 with deletion of the 3p21.3 region (3delp21.3) showed no effect on hTERT expression levels. These results provide direct evidence that hTERT suppressor gene(s) were retained in the 3p21.3 region, suggesting that the presence of regulatory factors that control telomerase enzyme activity may be involved in the development of OSCC.
