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PubMed1997

Mutations in MLH1 are more frequent than in MSH2 in sporadic colorectal cancers with microsatellite instability.

The microsatellite instability that is a feature of tumors in patients with hereditary nonpolyposis colorectal cancer (HNPCC) is a consequence of defective DNA mismatch repair. Mutations in the DNA mismatch repair genes MSH2 and MLH1 may account for up to 90% of HNPCC kindreds. Microsatellite instability is also seen in 10-16% of sporadic colorectal cancers. A limited number of MSH2 and MLH1 mutations have been described for sporadic colorectal cancers. In this study, we screened 12 primary sporadic colorectal cancers with microsatellite instability for mutations in MSH2 and MLH1 by using reverse transcription-polymerase chain reaction (RT-PCR) and single-strand-conformation-variant (SSCV) analysis. Eight mutations were identified in six tumors. One mutation in MLH1 was found to be present in the patient's germline DNA. Four tumors had somatic mutations in MLH1, and, in two of these tumors, two different mutations were identified. A single tumor had a somatic MSH2 mutation. Our observations suggest that MLH1 is mutated more frequently than MSH2 in sporadic colorectal cancers with microsatellite instability.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Molecular analysis of PinX1 in medulloblastomas.

Our group has previously demonstrated a high frequency of allelic loss on the short arm of chromosome 8 and identified a region of homozygous deletion of 1.41 Mb, flanked by D8S520 and D8S1130, on 8p23.1 in medulloblastomas, suggesting the presence of a tumor suppressor gene in this critical deletion region. The aim of our study was to investigate whether PinX1, a newly identified gene whose product is a potent inhibitor of telomerase, is the target gene in the homozygous deletion region identified in medulloblastomas. We assessed for alterations in gene sequence and transcript expression of PinX1, as well as the correlation between PinX1 expression and telomerase activity in a series of 52 medulloblastomas, 3 medulloblastoma cell lines (D283, D341 and Daoy) and 4 primitive neuroectodermal tumors (PNETs). Direct sequence analysis of all 7 exons and splice junctions of the PinX1 gene revealed no somatic mutations but 11 genetic polymorphisms. Transcript expression of PinX1, as evaluated by reverse transcription-polymerase chain reaction, in microdissected tumors and normal cerebellum showed 2 transcript variants, corresponding to the full-length form and an alternative spliced variant lacking exon 6, in all samples. This result indicated that PinX1 expression was not suppressed in medulloblastomas. Using the telomeric repeat amplification protocol (TRAP) assay, 13 of 19 (68%) medulloblastomas, 1 of 2 PNETs and all 3 cell lines showed telomerase activity. There is no significant correlation between PinX1 transcript expression and telomerase activity, but our results showed that telomerase activation is involved in medulloblastomas. Taken together, our results suggest that PinX1 does not play a major role in the oncogenesis of medulloblastomas.

Adolescent↗

PubMed2006

Mutations in DNA polymerase eta are not detected in squamous cell carcinoma of the skin.

The major etiological agent in skin cancer is exposure to UV-irradiation and the concomitant DNA damage. UV-induced DNA lesions, such as thymine dimers, block DNA synthesis by the major DNA polymerases and inhibit the progression of DNA replication. Bypass of thymine dimers and related lesions is dependent on the translesion polymerase DNA polymerase eta (Poleta). In the inherited disorder, xeroderma pigmentosum variant (XPV), inactivation of Poleta results in extreme sensitivity to UV light and a marked increase in the incidence of skin cancer. Here, we tested the hypothesis that somatic mutations and/or polymorphisms in the POLH gene that encodes Poleta are associated with the induction of UV-dependent skin cancers. We sequenced the exonic regions of the Poleta open reading frame in DNA from 17 paired samples of squamous cell skin carcinoma and adjacent histologically normal tissue. We analyzed approximately 120,000 nucleotides and detected no mutations in POLH in the tumors. However, we identified 6 different single-nucleotide polymorphisms, 3 of them previously undocumented, which were present in both the tumor and paired normal tissue. We conclude that neither mutations nor polymorphisms in the coding regions of POLH are required for the generation of human skin squamous cell carcinoma.

Adult↗

PubMed2005

Molecular evolution of herbicide resistance to phytoene desaturase inhibitors in Hydrilla verticillata and its potential use to generate herbicide-resistant crops.

Hydrilla [Hydrilla verticillata (Lf) Royle] is one of the most serious invasive aquatic weed problems in the USA. This plant possesses numerous mechanisms of vegetative reproduction that enable it to spread very rapidly. Management of this weed has been achieved by the systemic treatment of water bodies with the herbicide fluridone. At least three dioecious fluridone-resistant biotypes of hydrilla with two- to fivefold higher resistance to the herbicide than the wild-type have been identified. Resistance is the result of one of three independent somatic mutations at the arginine 304 codon of the gene encoding phytoene desaturase, the molecular target site of fluridone. The specific activities of the three purified phytoene desaturase variants are similar to the wild-type enzyme. The appearance of these herbicide-resistant biotypes may jeopardize the ability to control the spread of this non-indigenous species to other water bodies in the southern USA. The objective of this paper is to provide general information about the biology and physiology of this aquatic weed in relation to its recent development of resistance to the herbicide fluridone, and to discuss how this discovery might lead to a new generation of herbicide-resistant crops.

Crops, Agricultural↗

PubMed1987

Evidence for tetrameric structure of mammalian hypoxanthine phosphoribosyltransferase.

A fast electrophoretic variant of hypoxanthine phosphoribosyltransferase (HPRT) has been detected in Mus musculus bactrianus, a mouse subspecies from Middle Asia (USSR). The electrophoretic HPRT pattern yielded by hybrids between the somatic cell of LMTK- (deficient in thymidine kinase) and the splenocytes of a male of M. m. bactrianus was five-banded. The pattern obtained from the germ cells of the ovaries from 14.5-day-old embryos from laboratory CBA mice X M. m. bactrianus crosses was also composed of five bands. The hybrids between the somatic cells of human fibroblasts X LMTK- cells gave a three-banded electrophoretic HPRT pattern because the asymmetrical heteropolymeric isozymes are probably unstable. Taken together, all the evidence is in favor of a tetrameric structure of mammalian HPRT.

Animals↗

PubMed2000

Sloppier copier DNA polymerases involved in genome repair.

When chromosomal replication is impeded in the presence of DNA damage, members of a newly discovered UmuC/DinB/Rev1/Rad30 superfamily of procaryotic and eucaryotic DNA polymerases catalyze translesion synthesis at blocked replication forks. Although these polymerases share sequence elements essentially unrelated to the standard replication and repair enzymes, some of them (such as the SOS-induced Escherichia coli pol V) catalyze 'error-prone' translesion synthesis leading to large increases in mutation, whereas others (an example being the Xeroderma pigmentosum variant gene product XPV pol eta) carry out aberrant, yet nonmutagenic translesion synthesis. Ongoing studies of these low fidelity polymerases could provide new insights into the mechanism of somatic hypermutation, a key element in the immune response.

Animals↗

PubMed2005

Karyotypic abnormalities create discordance of germline genotype and cancer cell phenotypes.

The nature of mendelian inheritance assumes that all tissues in which a phenotype of interest is expressed have a uniform diploid karyotype, which is often not the case in cancer cells. Owing to nonrandom gains of chromosomes, trisomies are present in many cases of leukemia and other malignances. We used polymorphisms in the genes encoding thiopurine S-methyltransferase (TPMT), gamma-glutamyl hydrolase (GGH) and the reduced folate carrier (SLC19A1) to assess the nature of chromosomal acquisition and its influence on genotype-phenotype concordance in cancer cells. TPMT and GGH activities in somatic cells were concordant with germline genotypes, whereas activities in leukemia cells were determined by chromosomal number and whether the acquired chromosomes contained a wild-type or variant allele. Leukemia cells that had acquired an additional chromosome containing a wild-type TPMT or GGH allele had significantly lower accumulation of thioguanine nucleotides or methotrexate polyglutamates, respectively. Among these genes, there was a comparable number of acquired chromosomes with wild-type and variant alleles. Therefore, chromosomal gain can alter the concordance of germline genotype and cancer cell phenotypes, indicating that allele-specific quantitative genotyping may be required to define cancer pharmacogenomics unequivocally.

Adolescent↗

PubMed2005

Human intestinal IgA response is generated in the organized gut-associated lymphoid tissue but not in the lamina propria.

BACKGROUND & AIMS: The intestinal lamina propria has traditionally been viewed as the effector site of mucosal immune responses. However, this view has been challenged with the identification, in the murine lamina propria, of an in situ class switch DNA recombination pathway to IgA. In this study, we tested the hypothesis that in situ class switching occurs in the human lamina propria. METHODS: Immunohistochemistry was used to analyze tissue microenvironments and RT-PCR to look for molecular evidence of Ig class switching and to track clonally related cells of B lineage. RESULTS: We found no evidence of proliferation of either lamina propria CD20+ or CD19+ cells or evidence of activation-induced cytidine deaminase mRNA expression outside the organized gut-associated lymphoid tissue, although I alpha-C alpha immunoglobulin germ-line gene transcript expression could be identified in the lamina propria. We identified clonally related cells, including IgA and IgM isotype-switched variants, in multiple samples known to be free of activation-induced cytidine deaminase, organized lymphoid tissue, or cellular proliferation. For 4 groups of cells, the patterns of somatic mutations on the rearranged IgV(H)5 gene segment were more similar between cells from distant sites than from their immediate neighbors, implying dissemination of cells from a common set of precursors. CONCLUSIONS: Our data are inconsistent with a model in which precursors of human IgA-secreting plasma cells are induced or expanded in the lamina propria. The human lamina propria is therefore likely to solely be an effector site of intestinal secretory IgA responses that originate from the organized gut-associated lymphoid tissues.

Aged↗

PubMed1984

Fibroblasts from patients with hereditary cutaneous malignant melanoma are abnormally sensitive to the mutagenic effect of simulated sunlight and 4-nitroquinoline 1-oxide.

Because of a possible etiologic link between mutations and carcinogenesis, we compared fibroblasts derived from skin biopsies of several patients with hereditary cutaneous malignant melanoma and the dysplastic nevus syndrome for sensitivity to the mutagenic and/or cytotoxic effect of broad-spectrum simulated sunlight and of a UV mimetic carcinogen, 4-nitroquinoline 1-oxide (4NQO). The genetic marker was resistance to 6-thioguanine; loss of colony-forming ability was the assay for cytotoxicity. All five strains tested were more sensitive than normal to the killing effect of 4NQO (slopes of survival curves were 2- to 3-fold steeper), but only one strain was hypersensitive to killing by Sun Lamp radiation. Two strains were tested for mutagenicity. The response of each to the mutagenic action of these agents corresponded to its response to cell killing. Both strains were hypermutable after exposure to 4NQO, but only one showed a higher than normal frequency of mutants induced by simulated sunlight. The finding that nonmalignant fibroblasts from patients with a hereditary variant of malignant melanoma are abnormally susceptible to carcinogen-induced mutations suggests that hypersensitivity to mutagens contributes to risk of melanoma in patients. It also supports the somatic cell mutation hypothesis for the origin of cancer.

4-Nitroquinoline-1-oxide↗

PubMed2026

Tumor Mutational Concordance and Recurrence Timing in Hepatocellular Carcinoma.

INTRODUCTION: In hepatocellular carcinoma (HCC), intrahepatic recurrence includes true recurrence from clonal relapse and multicentric recurrence from de novo tumorigenesis. Recurrence timing is used to distinguish these types; however, its accuracy remains unclear. This study aimed to classify recurrent tumors based on somatic mutational concordance and assess the validity of recurrence timing. METHODS: Whole-exome sequencing was performed on paired primary and recurrent HCC tumors from 49 patients enrolled in a prospective institutional omics project. Tumors with ≥ 10 shared somatic mutations were classified as true recurrence. Clinicopathological features, recurrence timing, driver mutation patterns, and survival outcomes were compared between recurrence types. Mutational concordance was quantified using shared variant counts and the Jaccard similarity index. RESULTS: Of the 49 patients, 22 (44.9%) showed true recurrence and 27 (55.1%) had multicentric recurrence. Multicentric recurrence tumors harbored no shared variants or only a single shared variant with the primary tumor. True recurrence was associated with significantly higher concordance in histological differentiation and Edmondson-Steiner grading and greater retention of CTNNB1, TP53, ARID1A, and KEAP1 mutations. The number of shared variants (median: 115 vs. 0, and p < 0.001) and the Jaccard index (median: 0.44 vs. 0.00 and p < 0.001) were significantly higher in the true recurrence group. Recurrence timing was inconsistently correlated with mutational concordance, although a 3-year cutoff yielded significant separation. CONCLUSION: Recurrence timing alone insufficiently reflects clonal relationships. Genomic profiling offers a reliable framework for distinguishing between recurrence types and guiding HCC management.

clonal relapse↗

PubMed1999

Differences in allelic distribution of two polymorphisms in the VHL-associated gene CUL2 in pheochromocytoma patients without somatic CUL2 mutations.

Although the two major familial forms of pheochromocytomas, multiple endocrine neoplasia type 2 and von-Hippel-Lindau disease (VHL), have been associated with mutations of the RET and VHL genes, respectively, the molecular pathogenesis of sporadic pheochromocytomas is largely unknown. Recently, a putative tumor suppressor gene has been identified, CUL2, whose product has been shown to interact with the VHL tumor suppressor. To examine whether CUL2 plays a role in pheochromocytoma pathogenesis, we analyzed a series of 26 distinct tumor samples for mutations in the whole coding region of this gene. There were no somatic pathogenic mutations in CUL2, except for 1 sporadic tumor that had a hemizygous gene deletion. We also found 3 novel polymorphisms in the gene. One of these variants, IVS5-6C/T, as well as another previously described one, c.2057G/A, were overrepresented among the pheochromocytoma patients compared to that in a control population (P < 0.005 and P < 0.01, respectively). Although our findings suggest that CUL2 does not play a major role in the pathogenesis of pheochromocytomas, it is still unknown whether epigenetic mechanisms are involved in its inactivation in VHL-associated tumors. Furthermore, the potential role for the overrepresented alleles in the pheochromocytoma group requires further investigation.

Adolescent↗

PubMed2006

Analysis of the soluble isoforms of HLA-G mRNAs and proteins.

The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.

Enzyme-Linked Immunosorbent Assay↗

PubMed2004

The germinal center response.

The germinal center reaction is the foundation of T-cell-dependent humoral responses. Antigen-specific B cells recruited into germinal centers undergo a complex cellular program that allows for extensive expansion, isotype switching, somatic hypermutation, and differentiation into antibody-forming cells and memory cells. Importantly, the germinal center environment filters the repertoire of differentiating B cells such that high affinity variants are preferentially selected while low affinity or self-reactive clones are eliminated by apoptosis. The present article reviews the many processes that govern germinal center B-cell differentiation, as well as the cellular and molecular elements necessary to initiate and sustain a germinal center response. The major histologic features of the germinal center are also discussed, as well as the current dominant models of the germinal center reaction in humans and mice. Finally, a new model of murine B-cell differentiation is described on the basis of a multiparameter flow cytometric kinetic analysis of germinal center B cells.

Animals↗

PubMed1990

Congenital T-cell receptor immunodeficiencies in man.

The T-cell receptor (TCR) for antigen is a complex consisting of at least seven polypeptides chains. Two chains form the clonotypic heterodimer, which determines antigen-specificity. The other five constitute the monomorphic CD3 components, which are thought to be involved in signal transduction. We have reported a familial defect in the surface expression of the TCR/CD3 complex on otherwise phenotypically normal T lymphocytes. As a consequence of the surface defect, an impaired T-lymphocyte activation through the TCR and CD3 complex by both antigens and mitogens is observed in this type of immunodeficiency (ID) for which we propose the name TCR ID. In contrast, normal proliferative responses were recorded to TCR-independent activation or proliferation signals (i.e: anti-CD2, phorbol esters and IL-2). We believe that other ID with similar clinical and immunological characteristics, described before the general availability of monoclonal antibodies, may have also been TCR ID. Severe and mild clinical phenotypes of the disease exist which correlate with differences in the levels of TCR surface expression and of T-cell function in vitro. The biochemical basis of the defect in one of the cases thus far studied is an impaired association of CD3-zeta chain with the other chains of the complex. This defect prevented the maturation and transport of the incomplete complex to the cell surface. Due to the multi-subunit nature of the TCR/CD3 complex it is possible that other biochemical defects in TCR assembly may also give rise to TCR ID. The description of mild as well as a severe phenotype in TCR ID points to a threshold effect of TCR/CD3 expression necessary for normal T cell function in vivo since mild TCR ID is clinically asymptomatic. In contrast, severe TCR ID behaves as a clinical SCID, with clear autoimmune features and profound lymphoid tissue depletion. TCR ID and other naturally occurring SCID variants may shed light on fundamental questions of the development, function and biochemistry of the in vivo immune system. Their study and characterization also opens the door to somatic gene therapy in this kind of disorder, which is an obvious goal in ID research.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1997

Androgen receptor variants with short glutamine or glycine repeats may identify unique subpopulations of men with prostate cancer.

The androgen receptor (AR) contains glutamine (CAG) and glycine (GGC) repeats that are each polymorphic in length. We screened clinically localized prostate cancers for somatic mutations in the length of the CAG and GGC repeats in the AR gene and characterized the length of these repeats in the germ-line AR gene. Somatic mutations were rare, and the range of germ-line repeat lengths in men with prostate cancer was within the range of normal in the general population. Most allele frequencies in Caucasian men with clinical prostate cancer were remarkably comparable to those in the general Caucasian population. However, a subpopulation of the men with clinical prostate cancer had a substantially higher frequency of AR alleles with 16 or 17 CAGs (6 of 59 men, 10%) than did the general population (6 of 370 alleles, 1.6%), and a different subpopulation of the men with prostate cancer had a higher frequency of AR alleles with 12 or 13 GGCs (7 of 54 men, 13%) than did the general population (1 of 110 alleles, 0.9%). Of the men with prostate cancer who had an AR gene with 16 or 17 CAGs, 83% had lymph node-positive disease, despite the lack of clinical evidence of metastatic spread. This suggests that a short AR CAG allele may be a risk factor for the development of clinically unsuspected lymph node-positive prostate cancer among men undergoing radical prostatectomy and raises the question of whether this short repeat length played an active role in the development of aggressive prostate cancer. The odds of having a germ-line AR gene with a short CAG repeat (</=17 CAGs) were substantially higher in Caucasian men with lymph node-positive prostate cancer than in Caucasian men with lymph node-negative disease or in the general Caucasian population. The odds of having a short germ-line AR CAG were the same for men with lymph node-negative prostate cancer as for the general Caucasian population. The odds of having a germ-line AR gene with a short glycine repeat (</=14 GGCs) were substantially higher in men with prostate cancer than in the general population, but the frequency of alleles with a short GGC repeat was the same in men with lymph node-positive versus lymph node-negative disease. This suggests that a short GGC repeat may be a risk factor for the development of clinical prostate cancer, a hypothesis that needs to be tested in cohort and case-control studies.

Adenocarcinoma↗

PubMed2000

Organization and dynamics of satellite and telomere DNAs in Ascaris: implications for formation and programmed breakdown of compound chromosomes.

In the nematode genus Ascaris the germline genome contains considerable amounts of extra DNA, which is discarded from the somatic founder blastomeres during early cleavage. In Parascaris univalens the haploid germline genome is contained in one large compound chromosome, which consists of a euchromatic region containing the somatic genome flanked by large blocks of heterochromatin. Fluorescence in situ hybridization of fractions of the germline-limited satellite DNA revealed two highly repeated sequence families establishing the entire heterochromatin (HET blocks). The repeats, a pentanucleotide, TTGCA, and a decanucleotide, TTTGTGCGTG, constitute separate segments of the HET blocks. The blocks are polymorphic in length and, hence, in copy number of the repeats, and the arrangement of the segments. The numerous sequence variants of both repeats display a disperse distribution. The type and rate of base substitutions within both repeat units depend on position. Prior to the elimination process in presomatic cells, termed chromatin diminution, the chromosomes undergo differential mitotic condensation. Interstitial 'chromatin linkers' flanking the prospective numerous somatic chromosomes remain entirely decondensed. The somatic chromosomes are released from the plurivalent chromosomes via excision of the linkers at onset of anaphase, followed by exclusion of the akinetic linker chromatin and HET blocks from the daughter nuclei. In Ascaris suum, the germline-limited satellite, which consists of one 123 bp repeat, is scattered throughout the numerous chromosomes in small heterochromatic knobs of variable sizes, residing at chromosomal ends and/or intercalary positions. The programmed breakage, which appears to proceed in a similar manner to that in P. univalens, results in the loss of all heterochromatic knobs, accompanied by an increase in chromosome number. In both species, all germline chromosomes are capped by tracts of TTAGGC repeats. In P. univalens, such telomeric tracts also occur at the termini of the euchromatic intercalary regions. Upon diminution all telomeric tracts are discarded. De novo telomere addition occurs in all somatic cell lineages of both species. The presented data shed light on the evolutionary history of chromosome aggregation and satellite DNA formation, and putative mechanisms involved in the process of site-directed breakage to reestablish stable somatic chromosomes.

Animals↗

PubMed2002

Splice variants but not mutations of DNA polymerase beta are common in bladder cancer.

DNA polymerase beta (POLbeta) is a highly conserved protein that functions in base excision repair. Loss of the POLbeta locus on chromosome 8p is a frequent event in bladder cancer, and loss of POLbeta function could hinder DNA repair leading to a mutator phenotype. Both point mutations and large intragenic deletions of POLbeta have been reported from analysis of various tumor cDNAs but not from genomic DNA. We noticed that the breakpoints of the presumed rearrangements were delineated by exon-exon junctions, which could instead be consistent with alternative splicing of POLbeta mRNA. We tested the hypothesis that the reported intragenic deletion were splice variants by screening genomic DNA of human bladder tumors, bladder cancer cell lines, and normal bladder tissues for mutations or deletions in exons 1-14, exon alpha, and the promoter region of POLbeta. We found no evidence of somatic mutations or deletions in our sample set, although two polymorphisms were identified. Examination of cDNA from a subset of the original sample set revealed that truncated forms of POLbeta were surprisingly common. Forty-eight of 89 (54%) sequenced cDNA clones had large deletions, each beginning and/or ending exactly at exon-exon junctions. Because these deletions occur at exon-exon junctions and are seen in cDNA but not genomic DNA, they are consistent with alternative mRNA splicing. We describe 12 different splicing events occurring in 18 different combinations. Loss of exon 2 was the most frequent, being found in 42 of 49 (86%) of the variant sequenced clones. The splice variants appear to be somewhat more common and variable in bladder cancer cell lines and tumor tissues but occur at a high frequency in normal bladder tissues as well. We examine alternative splicing in terms of the information content of splice donor and acceptor site sequences, and discuss possible explanations for the predominant splicing event, the loss of exon 2.

Alternative Splicing↗

PubMed1984

Possible epigenetic mechanisms of tumor progression: induction of high-frequency heritable but phenotypically unstable changes in the tumorigenic and metastatic properties of tumor cell populations by 5-azacytidine treatment.

Treatment of a variety of highly tumorigenic mouse lines in vitro with chemical mutagens, such as ethyl methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), can result in extraordinarily high frequencies (sometimes in excess of 90%) of strongly immunogenic clones unable to grow progressively in normal syngeneic hosts. These clones will, however, grow in immunosuppressed hosts and gradually regain tumorigenic ability in normal mice if maintained in long-term (several months-1 year) culture, i.e., they are often phenotypically unstable. These features--phenotypic drift and high frequency--make it unlikely that point mutations are the underlying mechanism involved in the generation of the variants. Results presented here demonstrate that these observations can be reproduced on the same tumor lines using 5-azacytidine--an analogue of cytidine which can be incorporated into DNA causing subsequent extensive hypomethylation of cytosine residues in the absence of any significant mutagenic effects. Furthermore, 5-azacytidine treatment of a nonmetastatic mouse mammary tumor led to the emergence of a small number of heritable but unstable tumor clones capable of spontaneous metastatic spread. Because it is known that DNA hypomethylation can lead to transcriptional activation of normally silent genes, that altered methylation patterns can be somatically replicated with a high but not perfect fidelity, and that mutagens can cause DNA hypomethylation, we propose that DNA hypomethylation followed by de novo methylation represents a plausible mechanism to account not only for the induction of the nontumorigenic variants but for a number of aspects of tumor progression and tumor heterogeneity, as well. In particular, we refer to heritable phenotypic alterations in tumor cell populations which occur at very high frequency but which are not necessarily stable over very long periods of time.

Animals↗