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Biomedical subjects

S B Baylin

Publications and source records attributed to S B Baylin.

At least 91 records · Page 5Linked to original sources
PubMed1997

Frequent aberrant methylation of p16INK4a in primary rat lung tumors.

The p16INK4a (p16) tumor suppressor gene is frequently inactivated by homozygous deletion or methylation of the 5' CpG island in cell lines derived from human non-small-cell lung cancers. However, the frequency of dysfunction in primary tumors appears to be significantly lower than that in cell lines. This discordance could result from the occurrence or selection of p16 dysfunction during cell culture. Alternatively, techniques commonly used to examine tumors for genetic and epigenetic alterations may not be sensitive enough to detect all dysfunctions within the heterogeneous cell population present in primary tumors. If p16 inactivation plays a central role in development of non-small-cell lung cancer, then the frequency of gene inactivation in primary tumors should parallel that observed in cell lines. The present investigation addressed this issue in primary rat lung tumors and corresponding derived cell lines. A further goal was to determine whether the aberrant p16 gene methylation seen in human tumors is a conserved event in this animal model. The rat p16 gene was cloned and sequenced, and the predicted amino acid sequence of its product found to be 62% homologous to the amino acid sequence of the human analog. Homozygous deletion accounted for loss of p16 expression in 8 of 20 cell lines, while methylation of the CpG island extending throughout exon 1 was observed in 9 of 20 cell lines. 2-Deoxy-5-azacytidine treatment of cell lines with aberrant methylation restored gene expression. The methylated phenotype seen in cell lines showed an absolute correlation with detection of methylation in primary tumors. Aberrant methylation was also detected in four of eight primary tumors in which the derived cell line contained a deletion in p16. These results substantiate the primary tumor as the origin for dysfunction of the p16 gene and implicate CpG island methylation as the major mechanism for inactivating this gene in the rat lung tumors examined. Furthermore, rat lung cancer appears to be an excellent model in which to investigate the mechanisms of de novo gene methylation and the role of p16 dysfunction in the progression of neoplasia.

Amino Acid Sequence↗
PubMed1997

Protein kinase C-beta 2 inhibits cycling and decreases c-myc-induced apoptosis in small cell lung cancer cells.

The overexpression of c-myc frequently accompanies the relapse of small cell lung cancer (SCLC) cells and contributes to the poor prognosis of this tumor. In this study, we confirm that transfected c-myc results in decreased homotypic cell aggregation and increased proliferative capacity of SCLC cells when nutrient conditions are adequate. We also find that c-myc contributes to apoptosis when cells are nutrient depleted, and flow cytometry suggests that this enhanced apoptosis is associated with a failure to halt cell cycling, consistent with the experience in other cell types. We previously found that protein kinase C-beta (PKC-beta) expression in NCI H209 (209) SCLC cells increases markedly with c-myc transfection (L. F. Barr et al., Cancer Res., 51: 5514-5519, 1991), and we hypothesized that PKC-beta may mediate some of the effects of c-myc in these cells. We test this hypothesis by transfection of rat PKC-beta 1 and bovine PKC-beta 2 isoforms into 209 cells before and after transfection with c-myc. PKC-beta 1 transfection has no effect on these cells. However, PKC-beta 2 expression has distinct phenotypic consequences. In the parental cells, PKC-beta 2 expression results in increased homotypic cell aggregation and a prolonged doubling time. Furthermore, PKC-beta 2 expression increases the fraction of these cells in G0-G1. In the cells which express a transfected c-myc gene, PKC-beta 2 expression improves the survival of cells in low serum by decreasing myc-induced apoptosis. This effect was associated with, and may be mediated by, a selection for cells in the G0-G1 fraction. We postulate that transfection of c-myc into SCLC cells may select for those expressing the PKC-beta 2 gene because this signal transduction event protects against myc-induced apoptosis.

Animals↗
PubMed1997

DNA methylation changes in hematologic malignancies: biologic and clinical implications.

DNA methylation changes are among the most common detectable abnormalities in human neoplasia. Hypermethylation within the promoters of selected genes appears to be especially common in all types of human hematopoietic neoplasms, and is usually associated with inactivation of the involved gene(s). Such hypermethylation-associated silencing of gene expression has been shown for several genes regulating the growth and differentiation of hematopoietic cells, including the estrogen receptor (ER) gene, P15, P16 and others. Hypermethylation within the promoters of some genes appear to be an early event in the pathogenesis of neoplasia (ER, P15), while other genes seem to become methylated during the progression of leukemias (HIC1, c-abl). The high prevalence of promoter methylation suggests that this molecular abnormality can be used to monitor disease activity during therapy. In addition, new technology allows the sensitive identification of gene hypermethylation in a background of normal cells, suggesting possible new strategies for the detection of minimal residual disease. Finally, reactivation of tumor-suppressor gene expression through pharmacologic inhibition of DNA methyltransferase and resultant DNA demethylation appears to be a promising new avenue of therapy in acute leukemia.

Blood Cells↗
PubMed1997

Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer.

The tumor suppressor gene CDKN2 (p16/MTS1) resides on chromosome 9p21 and encodes a 16 kDa inhibitor of the cyclin-dependent kinases. Inactivation of CDKN2 by homozygous deletion, point mutation, and recently described aberrant methylation in the 5' promoter region may increase progression through the cell cycle in tumors. In this study, we examine the CDKN2 gene for the presence of inactivating alterations in human prostate cancer. Sequence analysis of cell lines revealed no mutation in LNCaP, PC3, and TSU-PR1 and a missense mutation, GAC-->TAC (asp to tyr), in exon 2 of the DU145 cell line at codon 76. No mutations were identified in three primary prostate cancers or in seven lymph node metastases. Loss of heterozygosity (LOH) was analyzed by analysis of microsatellite markers in the vicinity of the CDKN2 gene. LOH was detected in 12 (20%) of 60 primary tumors at one or more loci and in 13 (46%) of 28 metastases. Methylation analysis of the CpG-rich promoter region revealed a dense methylation of CDKN2 in cell lines PC3, PPC1, and TSU-PR1, and this was found to correlate with a lack of mRNA expression by reverse transcription-polymerase chain reaction. A demethylating agent, 5-aza-2'-deoxycytidine, induced reexpression when cells were exposed in vitro. DU145 and LNCaP expressed the CDKN2 transcript and were unmethylated in the promoter region. Three of twenty-four (13%) primary prostate cancers and 1 of 12 metastatic tumors demonstrated promoter methylation. No normal prostate tissues were methylated at the CDKN2 gene promoter. One tumor was found to contain concomitant LOH and promoter methylation indicative of biallelic inactivation. A comprehensive analysis of CDKN2 in prostate cancer reveals that point mutations are infrequent, but gene deletion and methylation combine to inactivate CDKN2 in a subset of tumors. Moreover, alterations in this gene may represent a late event in prostate cancer progression.

Aged↗
PubMed1997

Tissue-specific expression of human achaete-scute homologue-1 in neuroendocrine tumors: transcriptional regulation by dual inhibitory regions.

Malignancies with neuroendocrine (NE) features such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC) are prototypic neoplasms arising from peripheral endocrine cells. The mechanisms that regulate the NE phenotype in these tumors and their cellular precursors are not well understood. However, a basic helix-loop-helix transcription factor that is homologous to Drosophila neural fate determination proteins may have a central role. Human achaete-scute homologue-1 (hASH1), a human homologue of the Drosophila achaete-scute complex, is highly expressed in MTC, SCLC, and pheochromocytomas. To determine what mechanisms allow constitutive expression of hASH1 in NE tumors, we cloned human genomic DNA fragments containing the hASH1 gene and characterized its promoter region. We show that hASH1 expression is restricted to NE cell lines by a transcriptionally regulated mechanism. Dual promoters initiate hASH1 transcription, with the predominant site being an evolutionarily conserved initiator (INR) element. Transient transfection studies provide evidence for a generalized enhancer region that has high activity in all cell lines tested. Restriction of hASH1 expression to NE tumor cells depends on two tissue-specific repressor regions, present in the proximal and distal (> 13.5 kb) 5'-flanking region. Understanding the mechanisms of tissue-specific control of hASH1 gene expression provides a useful model to explore regulatory cascades influencing both normal nervous system development and the NE phenotype of tumors such as MTC and SCLC.

Basic Helix-Loop-Helix Proteins↗
PubMed1996

New 5' regions of the murine and human genes for DNA (cytosine-5)-methyltransferase.

DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) maintain patterns of methylated cytosine residues in the mammalian genome; faithful maintenance of methylation patterns is required for normal development of mice, and aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. The organization of coding sequences at the 5'-end of the murine and human DNA methyltransferase genes was investigated, and the DNA methyltransferase open reading frame was found to be longer than previously suspected. Expression of the complete open reading frame by in vitro transcription-translation and by transfection of expression constructs into COS7 cells resulted in the production of an active DNA methyltransferase of the same apparent mass as the endogenous protein, while translation from the second in-frame ATG codon produced a slightly smaller but fully active protein. Characterization of mRNA 5' sequences and the intron-exon structure of the 5' region of the murine and human genes indicated that a previously described promoter element (Rouleau, J., Tanigawa, G., and Szyf, M. (1992) J. Biol. Chem. 267, 7368-7377) actually lies in an intron that is more than 5 kilobases downstream of the transcription start sites.

Amino Acid Sequence↗
PubMed1996

Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis.

We have previously linked aging, carcinogenesis, and de novo methylation within the promoter of the estrogen receptor (ER) gene in human colon. We now examine the dynamics of this process for the imprinted gene for insulin-like growth factor II (IGF2). In young individuals, the P2-4 promoters of IGF2 are methylated exclusively on the silenced maternal allele. During aging, this promoter methylation becomes more extensive and involves the originally unmethylated allele. Most adult human tumors, including colon, breast, lung, and leukemias, exhibit increased methylation at the P2-4 IGF2 promoters, suggesting further spreading during the neoplastic process. In tumors, this methylation is associated with diminished or absent IGF2 expression from the methylated P3 promoter but maintained expression from P1, an upstream promoter that is not contained within the IGF2 CpG island. Our results demonstrate a remarkable evolution of methylation patterns in the imprinted promoter of the IGF2 gene during aging and carcinogenesis, and provide further evidence for a potential link between aberrant methylation and diseases of aging.

Adolescent↗
PubMed1996

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

Antisense Elements (Genetics)↗
PubMed1996

Methylation of the estrogen receptor CpG island in lung tumors is related to the specific type of carcinogen exposure.

Promoter methylation has recently been shown to be an alternative to mutation in inactivating tumor suppressor genes in human neoplasia. Although specific carcinogen exposures have been associated with characteristic mutation patterns in genes, the factors that lead to promoter hypermethylation remain unknown. One gene target for inactivation through promoter methylation is the estrogen receptor (ER). The purpose of this investigation was to determine the methylation status of this gene in lung tumors from smokers and those who never smoked and in rodents exposed to specific environmental carcinogens. Promoter methylation at the ER locus was detected in 4 of 11 tumors from never-smokers (36.4%) and 7 of 35 tumors from smokers (20%, P < 0.001). Lung tumors induced by the tobacco-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone also had a low incidence (16.7%) of ER methylation. In marked contrast, spontaneous and plutonium-induced tumors had a very high (81.8%) incidence of ER methylation. X-ray-induced tumors had an intermediate frequency of ER methylation (38.1%). The presence of ER methylation was associated with absent ER expression in rodent lung cancer cell lines. These results show for the first time that gene-specific promoter methylation can be modulated differentially depending on carcinogen exposure.

Animals↗
PubMed1996

Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer.

The association between increased DNA-methyltransferase (DNA-MTase) activity and tumor development suggest a fundamental role for this enzyme in the initiation and progression of cancer. A true functional role for DNA-MTase in the neoplastic process would be further substantiated if the target cells affected by the initiating carcinogen exhibit changes in enzyme activity. This hypothesis was addressed by examining DNA-MTase activity in alveolar type II (target) and Clara (nontarget) cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity was found only in the target alveolar type II cells of the susceptible A/J mouse and caused a marked increase in overall DNA methylation in these cells. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. Increased gene expression was also detected by RNA in situ hybridization in hypertrophic alveolar type II cells of carcinogen-treated A/J mice, indicating that elevated levels of expression may be a biomarker for premalignancy. Enzyme activity increased incrementally during lung cancer progression and coincided with increased expression of the DNA-MTase activity are strongly associated with neoplastic development and constitute a key step in carcinogenesis. The detection of premalignant lung disease through increased DNA-MTase expression and the possibility of blocking the deleterious effects of this change with specific inhibitors will offer new intervention strategies for lung cancer.

Animals↗
PubMed1996

The estrogen receptor CpG island is methylated in most hematopoietic neoplasms.

Estrogen appears to be a negative regulator of normal hematopoiesis. Chromosome 6q, which contains the estrogen receptor (ER) gene, is frequently altered in human hematopoietic neoplasms. The ER gene, which has growth and metastasis suppressor activity in many different cell types, is inactivated by promoter methylation in some ER-negative breast tumors and 100% of colorectal tumors. We now report that the promoter region of the ER gene is aberrantly methylated in 86% of human hematopoietic tumors, including 8 of 9 pediatric acute lymphocytic leukemia, 17 of 18 adult acute lymphocytic leukemia, 21 of 23 adult acute myelogenous leukemia, 3 of 6 chronic phase chronic myelogenous leukemia, 9 of 9 blast crisis chronic myelogenous leukemia and 5 of 8 lymphomas. This methylation event was also present in all nine leukemia cell lines examined, where it was associated with very low or absent ER expression. In addition, rat and mouse leukemia cell line also exhibited this change, indicating that ER CpG island methylation in leukemias is conserved among species. Our results suggest that ER CpG island methylation could be an important step in the genesis of human hematopoietic neoplasms and might be a useful molecular marker for monitoring the clinical status of these diseases.

Adult↗
PubMed1996

Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B.

The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms.

Adult↗
PubMed1996

Expression of prokaryotic HhaI DNA methyltransferase is transforming and lethal to NIH 3T3 cells.

In neoplastic cells, levels of DNA methyltransferase activity are often increased, and evidence is accruing to suggest an important role for this event in tumorigenesis. To evaluate this possibility further, and to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bacterial HhaI methyltransferase in cultured murine fibroblasts. This enzyme is a pure de novo DNA methyltransferase that methylates the internal C in the sequence GCGC. We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to the cells. However, surviving cell clones that express low levels of M. HhaI demonstrate increased tumorigenicity as assessed by soft agar cloning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0.4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 cells versus 0% for a mutant HhaI control) and tumorigenicity in nude mouse heterotransplants (75% for sense HhaI-transduced PA 317 cells versus 18.5% for antisense controls). DNA isolated from the clonogenic sense HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7-38.9) increase in methylcytosine content at GCGC sites. These findings suggest that eukaryotic cells tolerate a narrow window of increase de novo DNA methylating capacity, above which cell death occurs and within cell transformation results. Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.

3T3 Cells↗
PubMed1996

Differentiation of medullary thyroid cancer by C-Raf-1 silences expression of the neural transcription factor human achaete-scute homolog-1.

BACKGROUND: Human achaete-scute homolog-1 (hASH1), a fetal neural transcription factor, is highly expressed in neuroendocrine tumors such as medullary thyroid cancer (MTC). Although hASH1 probably plays a part in the growth and development of these tumors, its precise role and mechanism are unknown. METHODS: To further elucidate the function and regulation of hASH1 in neuroendocrine tumor differentiation, we used a model of MTC tumor differentiation mediated by the ras/raf-1 signaling pathway. The MTC TT cells alone or transduced with a beta-estradiol activatable raf-1 construct (TT: delta Raf-1:ER) were treated with beta-estradiol or carrier. Northern analysis and nuclear run-off assays were performed to determine the hASH1 messenger RNA (mRNA) levels and transcription rate, respectively. RESULTS: The TT: delta Raf-1:ER cells treated with beta-estradiol underwent marked biochemical and morphologic changes, including cell rounding, increase in calcitonin transcription, loss of RET proto-oncogene expression, and cessation of cell growth. During this differentiation process expression of hASH1 mRNA was silenced. Nuclear run-off experiments revealed that this decrease in steady-state hASH1 mRNA by raf-1 activation resulted predominantly from transcriptional inhibition. CONCLUSIONS: Silencing of hASH1 in parallel with loss of RET is associated with development of a mature C-cell differentiation pattern. Mechanisms leading to transcriptional silencing of hASH1 may be crucial in regulating the proliferative capacity or differentiation status of MTC. Downstream targets of hASH1 could play a role in C-cell proliferation and progression to MTC.

Basic Helix-Loop-Helix Proteins↗
PubMed1996

Hypermethylation of chromosome 17P locus D17S5 in human prostate tissue.

PURPOSE: Under normal conditions genomic CpG islands are not methylated. Hypermethylation of a CpG island in the 5' regulatory region of a gene has the capacity to silence gene transcription. Recently, hypermethylation of a CpG island at D17S5 on chromosome 17P13.3 has been shown to be a frequent tumor-specific event. When it has been observed, hypermethylation of D17S5 occurs solely in neoplastic tissues. Consequently, it has been hypothesized that hypermethylation of D17S5 may be an important carcinogenic event in the organs in which it occurs (colon, kidney, and brain). In this study we examine D17S5 hypermethylation in DNA from the prostate, a gland which is unique in that it undergoes hyperplastic or neoplastic growth or both in virtually all aging men. MATERIALS AND METHODS: The methylation sensitive restriction enzyme Notl, a cDNA probe specific for the D17S5 locus, and Southern blotting were used to assay for hypermethylation of D17S5 in DNA derived from normal, benign hyperplastic and malignant prostate tissues. RESULTS: We find that methylation of Notl restriction sites at D17S5 is a very common occurrence in prostate cancers (25 of 26 cases examined). Surprisingly, we found that methylation of these sites at D17S5 also occurred in histologically normal prostate and benign hyperplastic (BPH) tissue from glands which both did and did not contain cancer. In contrast, seminal vesicle, an androgen-dependent male sex accessory tissue that rarely undergoes pathological overgrowth, was devoid of hypermethylation at this locus. CONCLUSIONS. These data demonstrate that hypermethylation of D17S5 is a tissue-specific event in prostate DNA, and we hypothesize that methylation of this and/or related loci may play a role in the extreme predilection of this gland to neoplastic growth.

Base Sequence↗
PubMed1996

RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas.

An activated ras oncogene induces a program of differentiation in the human medullary thyroid cancer cell line TT. This differentiation process is accompanied by a marked increase in the transcription of the human calcitonin (CT) gene. We have localized a unique Ras-responsive transcriptional element (RRE) in the CT gene promoter. DNase I protection indicates two domains of protein-DNA interaction, and each domain separately can confer Ras-mediated transcriptional inducibility. This bipartite RRE was also found to be Raf responsive. By affinity screening, we have cloned a cDNA coding for a zinc finger transcription factor (RREB-1) that binds to the distal RRE. The consensus binding site for this factor is CCCCAAACCACCCC. RREB-1 is expressed ubiquitously in human tissues outside the adult brain. Overexpression of RREB-1 protein in TT cells confers the ability to mediate increased transactivation of the CT gene promoter-reporter construct during Ras- or Raf-induced differentiation. These data suggest that RREB-1 may play a role in Ras and Raf signal transduction in medullary thyroid cancer and other cells.

Adult↗
PubMed1996

De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.

Base Sequence↗