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S B Baylin

Publications and source records attributed to S B Baylin.

At least 127 records · Page 7Linked to original sources
PubMed1994

Stabilization of DNA methyltransferase levels and CpG island hypermethylation precede SV40-induced immortalization of human fibroblasts.

De novo methylation of normally unmethylated CpG islands and increased expression of DNA (cytosine-5)-methyltransferase (DNA MTase) are common characteristics of immortalized cell lines and human tumors. To examine the acquisition of these properties with respect to cellular immortalization, we studied CpG island methylation and DNA MTase expression in aging normal human fibroblasts and their SV40-infected preimmortal (precrisis) and immortal (postcrisis) derivatives. The levels of DNA MTase enzyme activity decreased by 50% as normal fibroblasts were cultured to senescence. By contrast, DNA MTase activity did not decrease in SV40-infected pre- or postcrisis cells but remained similar to that of young fibroblasts and 2-4-fold higher than that of senescent fibroblasts. DNA MTase mRNA levels paralleled those of enzyme activity. Several loci were examined to determine the relationship between the dynamics of DNA MTase expression and the appearance of de novo CpG island methylation. Ten CpG island loci examined were unmethylated in normal young fibroblasts. By contrast, four of these loci (the CALC1, MyoD, and IGF-2 genes on chromosome 11p and the estrogen receptor gene on chromosome 6q) were de novo methylated in fully immortalized, postcrisis cells. Two of these four were actually methylated in extended life span precrisis cells, and one, the estrogen receptor locus, exhibited de novo methylation with aging in normal fibroblasts. The data indicate that an ability to maintain DNA MTase levels is acquired with SV40-induced escape from senescence. Furthermore, aberrant CpG island methylation can be established prior to immortalization, either as a function of normal aging or in response to SV40-induced escape from senescence.

Blotting, Northern↗
PubMed1994

Molecular abnormalities in tumors associated with multiple endocrine neoplasia type 2.

The tumors of the MEN 2 syndromes, medullary thyroid carcinoma and pheochromocytoma, undergo defined stages of tumor progression. During these stages, several molecular abnormalities develop. These include abnormalities in growth, differentiation, and biochemistry. Recently, the germ-line abnormality in MEN 2A and familial medullary thyroid carcinoma has been identified in the ret gene. This article discusses possible molecular mechanisms for these abnormalities and attempts to place them in the context of the biology of the normal progenitor cells.

Adrenal Gland Neoplasms↗
PubMed1993

Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells.

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility.

3T3 Cells↗
PubMed1993

Increased cytosine DNA-methyltransferase activity during colon cancer progression.

BACKGROUND: Molecular changes during progressive stages of colon cancer and other human tumors commonly involve altered regulation of DNA methylation. These changes include overall genomic hypomethylation, regional hypermethylation, and increased levels of messenger RNA (mRNA) for cytosine DNA-methyltransferase (DNA-MTase), the enzyme that catalyzes DNA methylation at CpG (cytosine-phospho-guanine) sites. This increase in DNA-MTase transcripts (mRNA), if accompanied by increased DNA-MTase activity, could play a role in the abnormal DNA methylation patterns that appear early in colon tumor progression. PURPOSE: We sought to determine whether increased DNA-MTase mRNA levels during colon cancer progression are associated with increased cellular DNA-MTase enzymatic activity. METHODS: We adapted a microassay for DNA-MTase and used it to measure activity in human colon carcinoma and in colon mucosa of normal control subjects and of patients with colon cancer or with familial adenomatous polyposis (FAP), which is a risk factor for colon cancer. Steady-state DNA-MTase gene transcripts were measured by a reverse transcriptase polymerase chain reaction assay. To compare DNA-MTase activity with mRNA levels, we determined both variables simultaneously for one colon cancer specimen, its adjacent mucosa, and the colon mucosa of a control patient and compared the values. RESULTS: Compared with DNA-MTase activity in mucosa from normal control subjects, activity was elevated 1.4-fold in FAP mucosa, 1.6-fold in the uninvolved mucosa of patients with cancer, and 5.4-fold in the cancer specimens. All these differences were statistically significant. Fourteen of 15 cancer samples and 47% of the uninvolved adjacent mucosa samples had values that were higher than the highest value in normal mucosa. In one patient who had both a benign adenomatous polyp and a malignant adenocarcinoma, increasing DNA-MTase activity was observed at each stage of tumor progression. CONCLUSION: These results demonstrate that an increased DNA methylation capacity accompanies the increase in DNA-MTase transcripts observed during progressive stages of colon cancer. IMPLICATION: Further studies are needed to determine whether this abnormal methylation capacity plays a role in establishing the abnormal DNA methylation patterns seen in human malignancies.

Adenocarcinoma↗
PubMed1993

Identification of a human achaete-scute homolog highly expressed in neuroendocrine tumors.

Basic helix-loop-helix transcription factors of the achaete-scute family are instrumental in Drosophila neurosensory development and are candidate regulators of development in the mammalian central nervous system and neural crest. We report the isolation and initial characterization of a human achaete-scute homolog that is highly expressed in two neuroendocrine cancers, medullary thyroid cancer (MTC) and small cell lung cancer (SCLC). The human gene, which we have termed human achaete-scute homology 1 (hASH1), was cloned from a human MTC cDNA library. It encodes a predicted protein of 238 aa that is 95% homologous to mammalian achaete-scute homolog (MASH) 1, a rodent basic helix-loop-helix factor. The 57-residue basic helix-loop-helix domain is identical to that in the rodent gene, and the basic and helical regions, excluding the loop, are 72-80% identical to Drosophila achaete-scute family members. The proximal coding region of the hASH1 cDNA contains a striking 14-copy repeat of the triplet CAG that exhibits polymorphism in human genomic DNA. Thus, hASH1 is a candidate locus for disease-causing mutations via triplet repeat amplification. Analysis of rodent-human somatic cell hybrids permitted assignment of hASH1 to human chromosome 12. Northern blots revealed hASH1 transcripts in RNA from a human MTC cell line, two fresh MTC tumors, fetal brain, and three lines of human SCLC. In contrast, cultured lines of non-SCLC lung cancers and a panel of normal adult human tissues showed no detectable hASH1 transcripts. Expression of hASH1 may provide a useful marker for cancers with neuroendocrine features and may contribute to the differentiation and growth regulation of these cells.

Adrenal Gland Neoplasms↗
PubMed1993

DNA hypermethylation is associated with 17p allelic loss in neural tumors.

It has long been debated whether the accumulation of allelic losses in tumors involves the selection of cells which have stochastically lost chromosomal regions or whether there is, inherent to the neoplastic state, a process which predisposes to genetic instability. Changes in DNA methylation are commonly seen in human tumors and can alter chromosome structure. We now have examined specific types of primary neural tumors which allow us to determine relationships between abnormal DNA hypermethylation and allelic loss. In primary brain tumors which frequently lose chromosome 17p (30-50%) even in the earliest stages, we now show that 84% (21 of 25) exhibit hypermethylation at locus D17S5, on 17p. However, in primary neuroblastomas, a tumor type which does not lose chromosome 17p, no regional hypermethylation is observed. These data suggest that on chromosome 17p, regional D17S5 hypermethylation constitutes a molecular change which is associated with genetic instability.

Alleles↗
PubMed1993

Regional DNA hypermethylation at D17S5 precedes 17p structural changes in the progression of renal tumors.

In a preceding paper for brain tumors, we demonstrate a tight association between regional hypermethylation at locus D17S5 of chromosome 17p and allelic loss of this chromosome. Because 17p allelic losses occur at the earliest stages of brain tumors, the exact temporal relationship between this event and the hypermethylation could not be elucidated. In renal cancers, two linked structural changes on chromosome 17p, allelic loss and p53 gene mutations, generally occur late in progression. We now show that D17S5 hypermethylation is tightly coupled to both of these genetic changes in late stage renal tumors. However, the methylation change is the only one of the 17p abnormalities which occurs at a high incidence in early-stage renal cancers (hypermethylation, 50%; 17p allelic loss, 13%; p53 mutations, 0%). Our results firmly suggest that D17S5 regional hypermethylation precedes the appearance of the consistent 17p genetic changes in renal cancers, suggesting that this event either marks, or may even cause, chromatin changes which predispose to genetic instability.

Alleles↗
PubMed1993

Altered chromosomal methylation patterns accompany oncogene-induced transformation of human bronchial epithelial cells.

Abnormal methylation of CpG island sequences on chromosomes 11p and 17p, and tumor phenotype-associated differential methylation of chromosome 3p loci have been described in human lung tumors (S.B. Baylin, J.W.M. Hoppener, A. de Bustros, P.H. Steenbergh, C.J.M. Lips, and B. D. Nelkin, Cancer Res., 46: 2917-2922, 1986; M. Makos, B.D. Nelkin, M. I. Lerman, F. Latif, B. Zbar, and S.B. Baylin, Proc. Natl. Acad. Sci. USA, 89: 1929-1933, 1992; A. de Bustros, B. D. Nelkin, A. Silverman, G. Ehrlich, B. Poiesz, and S. B. Baylin, Proc. Natl. Acad. Sci. USA, 85: 5693-5697, 1988). Using an in vitro model of lung tumor progression, we now show that these aberrant methylation patterns occur at different stages during cellular immortalization and oncogene-induced neoplastic transformation of normal human bronchial epithelial cells (NHBE). The CALC1 CpG island locus on chromosome 11p15.4 was essentially unmethylated in NHBE and simian virus 40 T-antigen immortalized NHBE (BEAS-2B cells) but became de novo methylated in 5 of 6 BEAS-2B derived cell lines that were transfected or infected with various oncogenes and in a spontaneously neoplastically transformed subline of BEAS-2B cells. By contrast, an additional CpG island locus, pYNZ22, at 17p13.3 became fully methylated following the immortalization of NHBE and was not further changed by oncogene-induced transformation. Finally, at a non-CpG island locus pYNZ86.1 on chromosome 3p14, different tumor phenotype-associated methylation patterns became apparent only after passage of the turmorigenic oncogene-transformed bronchial epithelial cell lines in athymic nude mice. Whereas cell lines derived from tumors with a non-small cell lung carcinoma-like phenotype were significantly hypomethylated relative to their parental cell lines, a cell line derived from a tumor with a more small cell lung carcinoma-like phenotype retained the methylation status of its parental cell line. The data indicate that altered DNA methylation patterns, including the de novo methylation of normally unmethylated CpG island sequences and demethylation of nonisland sequences, arise at different stages during immortalization and oncogene-induced neoplastic transformation of bronchial epithelial cells. These findings suggest that DNA methylation abnormalities accompany, or may play a role in, the genetic changes that occur during lung tumor progression.

Base Sequence↗
PubMed1993

Management of pheochromocytomas in patients with multiple endocrine neoplasia type 2 syndromes.

OBJECTIVE: The authors sought to determine the optimal surgical management of pheochromocytomas that develop in patients with multiple endocrine neoplasia (MEN) type 2 syndromes. SUMMARY BACKGROUND DATA: The performance of empirical bilateral adrenalectomy in patients with MEN 2A or MEN 2B, whether or not they have bilateral pheochromocytomas, is controversial. METHODS: The results of unilateral or bilateral adrenalectomy were studied in 58 patients (49 with MEN 2A and 9 with MEN 2B). Recurrence of disease was evaluated by measuring 24-hour urinary excretion rates of catecholamines and metabolites and by computed tomography (CT) scanning. RESULTS: The mean postoperative follow-up was 9.40 years. There was no operative mortality and malignant or extra-adrenal pheochromocytomas were not present. Twenty-three patients with a unilateral pheochromocytoma and a macroscopically normal contralateral gland underwent unilateral adrenalectomy. A pheochromocytoma developed in the remaining gland a mean of 11.87 years after the primary adrenalectomy in 12 (52%) patients. Conversely, 11 (48%) patients did not develop pheochromocytoma during a mean interval of 5.18 years. In the interval after unilateral adrenalectomy, no patient experienced hypertensive crises or other complications related to an undiagnosed pheochromocytoma. Ten (23%) of 43 patients having both adrenal glands removed (either at a single operation or sequentially) experienced at least one episode of acute adrenal insufficiency or Addisonian crisis, including one patient who died during a bout of influenza. CONCLUSIONS: Based on these data, the treatment of choice for patients with MEN 2A or MEN 2B and a unilateral pheochromocytoma is resection of only the involved gland. Substantial morbidity and significant mortality are associated with the Addisonian state after bilateral adrenalectomy.

Addison Disease↗
PubMed1993

Preoperative localization of occult medullary carcinoma of the thyroid gland with single-photon emission tomography dimercaptosuccinic acid.

BACKGROUND: Patients who undergo thyroidectomy for medullary carcinoma of the thyroid gland (MTC) often have elevations of postoperative serum calcitonin levels, which are indicative of metastatic or residual disease. It has been extremely difficult to localize tumor in these patients with standard diagnostic studies such as ultrasonography, computed tomography, or magnetic resonance imaging scans. Previous studies have suggested that planar technetium 99m (V) dimercaptosuccinic acid (DMSA) scintigraphic scans can localize MTC in these patients. We have recently increased the sensitivity of planar scintigraphic images by using single-photon emission tomography (SPECT). This study was performed to compare the sensitivity of planar DMSA scans with that of SPECT DMSA scans. METHODS: Two normal volunteers and three patients with occult MTC after previous total thyroidectomy underwent planar and SPECT DMSA scans. Each patient subsequently underwent surgical exploration based on the DMSA scans. RESULTS: Physiologic DMSA uptake was noted in the nasopharynx, axial skeleton, breast, liver, spleen, heart, kidneys, urinary bladder, great vessels, and skeletal muscles in both normal volunteers and patients with occult MTC. Planar DMSA scans and dynamic computed tomographic scans failed to localize MTC in any of these patients who had minimal disease. SPECT DMSA scans correctly localized cervical MTC in two of three patients, as proved by subsequent surgical resection. One patient who had a negative cervical exploration is presumed to have had a false-positive SPECT DMSA scan. CONCLUSIONS: SPECT DMSA scans appear to be a sensitive, safe, and noninvasive localization technique for patients with occult MTC who have undergone previous thyroidectomy.

Adult↗
PubMed1993

Pathobiology of the C-cells.

This brief review of the pathobiology of C-cells has stressed cellular and molecular aspects of MTC development. In the genetic forms of MTC, an alteration in one or more genes on chromosome 10, through an as yet unknown series of events, results in initial hyperproliferation of C-cells. Subsequent genetic steps, possibly at other chromosome loci, presumably result in selected clonal transformation of the hyperproliferative C-cells at risk for tumor development. Some of these same molecular events are probably operative in the development of sporadic MTC as well. Once MTC has developed, it has the potential to undergo tumor progression events which result in loss of C-cell differentiation. Studies in a culture model of these events have revealed that activation of signal transduction pathways, similar to those active in differentiation of other neural crest-derived cells, can restore differentiation features of normal C-cells to MTC. Continued identification of the molecular factors mediating this restoration should teach us much about the relationships between general neural crest differentiation and that of normal C-cells. It will also reveal much about the pathobiology of C-cells contributing to each step of MTC development.

Carcinoma↗
PubMed1992

Characterization of an endogenous RNA transcript with homology to the antisense strand of the human c-myc gene.

In addition to being regulated by a complex array of cis- and trans-acting factors, c-myc protooncogene expression may be modulated by antisense RNA transcripts. Our previous studies have determined that depletion of intracellular polyamines by alpha-difluoromethylornithine results in a marked decrease in the transcription of the human c-myc gene. Because of reports that antisense transcription occurs in the 5' and 3' regions of this gene, we used a genomic clone of the human c-myc gene to ascertain whether polyamine depletion might induce an antisense RNA transcript. These studies demonstrate that polyamine depletion of the human colon cancer cell line COLO 320 results in induction of an endogenous RNA transcript with high homology to the antisense strand of the second intervening sequence (PvuII-RsaI) of the c-myc gene. Furthermore, during such depletion, steady state levels of this transcript vary inversely to the sense direction c-myc RNA. RNase protection studies suggest that the antisense transcript may arise from a different gene locus than the c-myc gene. To further identify the origins of this RNA, a cDNA library was generated from size-selected RNA and screened with c-myc sequences. A 438-base pair cDNA was isolated with approximately 85% homology, to a 285-base region in the second intron of the c-myc gene. Computer homology analysis further reveals that a 120-base region within this cDNA also has approximately 85% homology to the antisense strands of a number of genes, including the growth-related genes, N-myc, p53, and thymidine kinase. These studies provide the initial characterization of an endogenous antisense RNA transcript which could influence cell growth by modulating the expression of c-myc and other genes.

Base Sequence↗
PubMed1992

Isolation and characterization of the cDNA encoding human DNA methyltransferase.

We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

Amino Acid Sequence↗
PubMed1992

Distinct hypermethylation patterns occur at altered chromosome loci in human lung and colon cancer.

Regional increases in DNA methylation occur in normally unmethylated cytosine-rich areas in neoplastic cells. These changes could potentially alter chromatin structure to inactivate gene transcription or generate DNA instability. We now show that, in human lung and colon cancer DNA, hypermethylation of such a region consistently occurs on chromosome 17p in an area that is frequently reduced to homozygosity in both tumor types. Over the progression stages of colon neoplasia, this methylation change increases in extent and precedes the allelic losses on 17p that are characteristic of colon carcinomas. We also show on chromosome 3p that regional hypermethylation may nonrandomly accompany chromosome changes in human neoplasia. Increased methylation is consistent in small-cell lung carcinoma DNA at two 3p loci that are constantly reduced to homozygosity in this tumor, but it is not seen in colon cancer DNA, in which these loci are infrequently structurally altered.

Carcinoma↗
PubMed1992

Consistent association of 1p loss of heterozygosity with pheochromocytomas from patients with multiple endocrine neoplasia type 2 syndromes.

Pheochromocytomas and medullary thyroid cancers (MTCs) are neuroendocrine tumors which arise sporadically or as part of the multiple endocrine neoplasia type 2 (MEN-2) hereditary syndromes. The most consistent molecular genetic abnormality which has been described in these tumors is loss of heterozygosity (LOH) of the short arm of chromosome 1 (1p). This finding is particularly interesting because the predisposition gene for the hereditary form of these tumors has been mapped to chromosome 10, but LOH on chromosome 10 in MEN-2 tumors is found rarely. We have used a battery of 1p DNA probes to elucidate the region of loss of 1p in 18 pheochromocytomas and 27 MTCs. Using restriction fragment length polymorphism analysis, we identified loss of all or a portion of 1p in 12 of 18 pheochromocytomas. 1p LOH was identified in nine of nine pheochromocytomas in MEN-2A and -2B patients, compared with only two of seven sporadic pheochromocytomas. We also found 1p LOH in one of two von Hippel-Lindau patients. LOH on 1p was noted in only three of 24 informative MTCs, and these were from patients with MEN-2A. In most of the pheochromocytomas, the entire short arm of chromosome 1p appears to have been lost; however, in three of the non-MEN pheochromocytomas and in three MEN-2A MTCs, the region of loss is smaller, allowing estimation of the smallest region of overlap. The combined data for MTCs and pheochromocytomas suggest that the smallest region of overlap of LOH is bounded by D1S15 (1pter-p22) and D1Z2 (1P36.3), excluding a region around MYCL (1p32). Although other regions of 1p should not be completely ruled out, the data suggest that this region may harbor a tumor suppressor gene or genes whose inactivation is important in the development of these tumors. Furthermore, the strong association between 1p LOH and the MEN-2 syndromes, especially in pheochromocytomas, suggests a relationship between the predisposition gene on chromosome 10 and the loss of the suppressor gene on 1p. Alternatively, other loci may be more important in sporadic disease.

Adrenal Gland Neoplasms↗
PubMed1992

Human calcitonin gene regulation by helix-loop-helix recognition sequences.

Human calcitonin (CT) gene transcription is regulated by proximal 5' flanking sequences which mediate cAMP-induced expression, and by a distal basal enhancer region. Using transient expression of CT-CAT constructs, we showed that the basal enhancer is active in a CT-producing small cell lung cancer cell line (DMS53) and the thyroid C cell derived tumor line, TT, but is inactive in non-CT-producing cell lines. In deletional and direct mutational analyses of the distal enhancer region, disruption of two elements resembling recognition sequences for the helix-loop-helix (HLH) family of transcriptional regulatory proteins resulted in a significant loss of basal transcriptional enhancer action. These results suggest that HLH recognition motifs may mediate a significant portion of constitutive CT gene transcriptional activity in these cells. Nuclear protein extracts from DMS53 cells formed specific binding complexes with oligonucleotides containing two of these candidate enhancer sequences. However, proteins capable of binding to these CT gene HLH consensus recognition sites were not restricted to CT-producing cells. We conclude that members of the HLH protein family, some expressed ubiquitously and some expressed or activated in a tissue-restricted fashion, may combine to enhance CT gene transcription in tumor cells of neuroendocrine derivation.

Base Sequence↗