p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3.

For several human tumour types, allelic loss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5' flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of G418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.

Mutation affecting the 12th amino acid of the c-Ha-ras oncogene product occurs infrequently in human cancer.

A point mutation alters the 12th amino acid of the c-Ha-ras oncogene product p21 in a human bladder cancer cell line. This is, at present, the only mutation known to result in a human transforming gene. This mutation may therefore represent a possible target for mutagenesis leading to carcinogenesis in humans. By means of restriction enzyme analysis, 29 human cancers, including 20 primary tumor tissues, derived from organs commonly exposed to environmental carcinogens, were tested for the presence of this mutation. None of ten primary bladder carcinomas exhibited the mutation; nor did nine colon carcinomas or ten carcinomas of the lung. Thus the point mutation affecting the 12th amino acid of the c-Ha-ras gene product, while a valuable model for carcinogenesis, does not appear to play a role in the development of most human epithelial cancers of the bladder, colon, or lung.

v-rasH induces non-small cell phenotype, with associated growth factors and receptors, in a small cell lung cancer cell line.

Small cell lung cancer (SCLC) tumor progression can involve partial or complete conversion to a more treatment-resistant non-small cell (NSCLC) phenotype. In a cell culture model of this phenomenon, we have previously demonstrated that insertion of the viral Harvey ras gene (v-Ha-ras) into SCLC cell lines with amplification and overexpression of the c-myc gene induced many NSCLC phenotypic features. We now report that the v-Ha-ras gene can also induce morphologic, biochemical, and growth characteristics consistent with the NSCLC phenotype in an N-myc amplified SCLC cell line, NCI-H249. We show that v-Ha-ras has novel effects on these cells, abrogating an SCLC-specific growth requirement for gastrin-releasing peptide, and inducing mRNA expression of three NSCLC-associated growth factors and receptors, platelet-derived growth factor B chain, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R). TGF-alpha secretion and EGF-R also appear, consistent with the induction of an autocrine loop previously shown to be growth stimulatory for NSCLC in culture. These data suggest that N-myc and v-Ha-ras represent functional classes of genes that may complement each other in bringing about the phenotypic alterations seen during SCLC tumor progression, and suggest that such alterations might include the appearance of growth factors and receptors of potential importance for the growth of the tumor and its surrounding stroma.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.

Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma.

Regulation of expression of the human calcitonin gene was found to differ between two tumor lines of different tissue origin, medullary thyroid carcinoma (TT line) and small-cell lung carcinoma (DMS53 line). Distal 5' DNA elements between -750 and -2000 exhibited a stronger basal activity in DMS53 than in TT cells, whereas proximal DNA sequences between -132 and -252 mediated a dramatic cyclic AMP response in TT but not DMS53 cells.

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.

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.

Transcriptional and posttranscriptional modulation of calcitonin gene expression by sodium n-butyrate in cultured human medullary thyroid carcinoma.

The TT cell line of human medullary thyroid carcinoma produces large quantities of calcitonin (CT) and calcitonin gene-related peptide (CGRP) mRNAs by alternative splicing of a primary CT gene transcript. We have previously shown that the relative levels of these mRNAs depend on the growth stages of the TT cells in culture and that these mRNAs can be increased acutely at the transcriptional level by phorbol esters (12-O-tetradecanoylphorbol-13-acetate) and the cyclic nucleotide, cyclic AMP. We show here that the naturally occurring fatty acid butyrate, unlike 12-O-tetradecanoylphorbol-13-acetate or cyclic AMP, has a delayed stimulatory effect on CT gene transcription, and also can modulate the posttranscriptional processing of RNA from this gene. Treatment of the TT cells with butyrate leads to a 5-fold increase in CT gene transcription after a lag period of 48 h and to a sustained decrease in the calcitonin gene-related peptide to CT mRNA ratio throughout the growth curve of these cells. In addition to its effects on CT gene expression, butyrate also decreases cellular proliferation and c-myc expression in the TT cells. These changes suggest that butyrate induces cultured human medullary thyroid carcinoma cells to acquire in vitro properties more consistent with the differentiated phenotype of the mature thyroid C-cell which is characterized by a low calcitonin gene-related peptide to CT ratio.

Ret gene silencing is associated with Raf-1-induced medullary thyroid carcinoma cell differentiation.

Mutations in the ret proto-oncogene constitute the germ line defect in patients with inherited forms of medullary thyroid carcinoma (MTC) and are also present in tumor DNA from a subset of patients with sporadic forms of MTC. We now show that the TT cell line of human MTC can be induced within 48 h to resemble mature C cell differentiation by activation of the raf-1 signal transduction pathway. Within this time period, expression of both the mutant and wild-type ret gene alleles, present in these cells, are silenced at the mRNA and protein levels. This definition of a signal transduction pathway that can regulate ret gene expression, and of the position of ret gene expression in endocrine differentiation, should help clarify the precise role of this gene in normal neuroendocrine development and in the formation of MTC.

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.

Changes in calcitonin gene RNA processing during growth of a human medullary thyroid carcinoma cell line.

The ratios of calcitonin (CT) to calcitonin gene-related peptide (CGRP) mRNA, both generated by alternative RNA processing from the same primary RNA transcript, are shown by Northern blotting of cytoplasmic RNA to vary as a function of growth in a human medullary thyroid carcinoma cell line (TT). Upon initial seeding, CT mRNA levels are relatively high, and CGRP mRNA levels are relatively low. During the early logarithmic growth phase, CGRP mRNA levels rise severalfold, while CT mRNA levels change only slightly. As the cells approach confluence, both CT and CGRP mRNA levels rise. Subsequently, CGRP mRNA levels fall substantially in postconfluent cells, while CT mRNA levels remain high. By actinomycin D blocking of nascent transcription, we have shown that these growth-related, reversible changes in the ratio of CT to CGRP mRNA are not due to changes in mRNA stability. Our data rather suggest that TT cells reversibly alter alternative RNA-processing patterns dependent upon growth conditions in vitro, such that CT mRNA is lowest and CGRP mRNA is highest during rapid growth. The mechanisms underlying this RNA-processing alteration may play a role in certain patients with aggressive forms of medullary thyroid carcinoma, in whom a decrease or loss of CT levels heralds a poor prognosis.

c-myc gene-induced alterations in protein kinase C expression: a possible mechanism facilitating myc-ras gene complementation.

The mechanism(s) by which the c-myc nuclear protein and the membrane-associated ras protein interact to mediate phenotypic changes is unknown. We now find that c-mcy gene expression is associated with alterations in the principal signal transduction pathway through which the ras protein is thought to function. We studied the transcript and protein expression of protein kinase C (PKC) isoforms in a culture line of human small cell lung cancer cells (NCI H209) in which expression of inserted c-myc and Ha-ras genes together, but not alone, causes a transition to a large cell phenotype. In control H209 cells, at the transcript and cell membrane protein levels, PKC-alpha is the dominant PKC species. In this cell line, the expression of an exogenous c-myc gene, but not of a viral Ha-ras gene, causes a 5- to 10-fold increase in the PKC-beta isoform transcript and protein. The insertion of ras into the exogenous myc-expressing 209 cells, in addition to causing phenotypic transition, results in the translocation of the PKC-beta protein from the cytosol to the membrane fraction and a decrease in membrane-associated PKC-alpha. Concomitant with these changes, the increased PKC isoform transcript levels induced by myc alone are completely reversed. These observations suggest that a complex set of PKC transcript and protein alterations, most prominently involving an increased PKC-beta protein level in the cell membrane, a decrease in PKC-alpha protein, and a decrease in all PKC isoform transcripts, may represent a fundamental event(s) for c-myc collaboration with Ha-ras to alter cell phenotype.

Constitutive achaete-scute homologue-1 promotes airway dysplasia and lung neuroendocrine tumors in transgenic mice.

The transcription factor achaete-scute homologue-1 (ASH1) is essential for neural differentiation during fetal development and is a cardinal feature of neuroendocrine (NE) tumors such as small cell lung cancer. To explore the potential of ASH1 to promote NE differentiation and tumorigenesis in the lung, we constitutively expressed the factor in nonendocrine airway epithelial cells using transgenic mice. Progressive airway hyperplasia and metaplasia developed beginning at 3 weeks of life. ASH1 potently enhanced the tumorigenic effect of SV40 large T antigen in airway epithelium. These doubly transgenic animals developed massive NE lung tumors, implying that ASH1 may cooperate with defects in p53, pRb, or related pathways in promoting NE lung carcinogenesis.

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.

DNA methylation patterns of the calcitonin gene in human lung cancers and lymphomas.

Generalized hypomethylation of the genome and of specific genes has been described in human tumors. We now report that in human lung cancers, especially in the most aggressive form, small cell lung carcinoma, and in lymphomas, the 5'-region of the calcitonin (CT) gene exhibits methylation of increased numbers of CCGG sites in comparison with normal adult tissues. These unusual methylation patterns are found much less frequently in other tumor types examined. In the spectrum of the four major types of lung cancer (small cell, adeno-, squamous, and large cell carcinomas), the frequency of occurrence of hypermethylation in the 5'-region of the CT gene parallels that for presence of the neuroendocrine related biochemistry which characterizes small cell lung carcinoma. In medullary thyroid carcinoma, a tumor which expresses high levels of CT gene mRNA, the 5'-region of the CT gene is hypomethylated. Our findings provide a potential new molecular marker for two important human cancers (lung cancer and lymphomas) and suggest that there is a close relationship between abnormal CT gene methylation and developmental events for these tumors.

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.

Notch signaling induces cell cycle arrest in small cell lung cancer cells.

Among the various forms of human lung cancer, small cell lung cancer (SCLC) exhibits a characteristic neuroendocrine (NE) phenotype. Neural and NE differentiation in SCLC depend, in part, on the action of the basic-helix-loop-helix (bHLH) transcription factor human achaete-scute homologue-1 (hASH1). In nervous system development, the Notch signaling pathway is a critical negative regulator of bHLH factors, including hASH1, controlling cell fate commitment and differentiation. To characterize Notch pathway function in SCLC, we explored the consequences of constitutively active Notch signaling in cultured SCLC cells. Recombinant adenoviruses were used to overexpress active forms of Notch1, Notch2, or the Notch effector protein human hairy enhancer of split-1 (HES1) in DMS53 and NCI-H209 SCLC cells. Notch proteins, but not HES1 or control adenoviruses, caused a profound growth arrest, associated with a G1 cell cycle block. We found up-regulation of p21(waf1/cip1) and p27kip1 in concert with the cell cycle changes. Active Notch proteins also led to dramatic reduction in hASH1 expression, as well as marked activation of phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2, findings that have been shown to be associated with cell cycle arrest in SCLC cells. These data suggest that the previously described function of Notch proteins as proto-oncogenes is highly context-dependent. Notch activation, in the setting of a highly proliferative hASH1-dependent NE neoplasm, can be associated with growth arrest and apparent reduction in neoplastic potential.

Low incidence of loss of chromosome 10 in sporadic and hereditary human medullary thyroid carcinoma.

Genetic linkage has been recently documented between a centromeric region of chromosome 10 and familial multiple endocrine neoplasia type II (MEN II). This syndrome consists of initial thyroid C-cell and adrenal chromaffin cell hyperplasia which result in multifocal medullary thyroid carcinomas and bilateral adrenal pheochromocytomas. Other hereditary cancers, such as retinoblastoma, appear to result from a series of genetic events involving, first the inheritance of a germ line abnormality, and subsequent loss of chromosome loci opposite this initial defect. In these cancers, this loss of the normal alleles in both familial and sporadic cases, is frequently manifest as a reduction to homozygosity for polymorphic DNA markers near the involved locus. It might then be expected that chromosome 10 regions would be lost with high frequency in tumor DNA from patients with MEN II and sporadic medullary thyroid carcinoma (MTC). We now demonstrate that only two of 16 MTC tumors studied by analysis of restriction fragment length polymorphisms for multiple regions of the short and long arms of chromosome 10 showed loci reduced to homozygosity. One of these tumors was from a patient with MEN II and the other from a patient with nonfamilial MTC. Importantly, no such chromosome 10 changes were noted in pheochromocytomas from the patient with MEN II or his sister. These findings strongly suggest that the sequence of genetic events for familial MTC is either different from that for retinoblastoma or that loss of normal alleles opposite the germ line genetic defect occurs by mechanisms other than gross loss of chromosomal material in MTC. A model is proposed suggesting that the mechanism involving loss of alleles opposite one another is operative in hereditary tumors, such as retinoblastoma, which do not arise within a setting of initial polyclonal cellular hyperplasia. In contrast, in tumors such as familial MTC and polyposis coli which arise as individual clones of neoplastic cells from a setting of preexistent polyclonal hyperplasia, the first genetic event may underlie hyperplasia, and additional events, frequently at other chromosomal loci, may cause individual clonal neoplasms.

BARX2 induces cadherin 6 expression and is a functional suppressor of ovarian cancer progression.

The human homeobox BARX2 is located at 11q24-q25, within a minimal region associated with frequent loss of heterozygosity and adverse survival in epithelial ovarian cancer. BARX2 is a transcription factor that regulates transcription of specific cell adhesion molecules in the mouse. We show that BARX2 and cadherin 6 are expressed in normal human ovarian surface epithelium. BARX2 and cadherin 6 both have significantly lower expression in a clinical sample of endometrioid and clear cell ovarian cancers, as compared with serous or mixed mesodermal tumors. In a series of ovarian cancer cell lines, BARX2 expression showed a significant direct correlation with cadherin 6 expression. In OAW42, an ovarian cancer cell line that does not endogenously express BARX2, in vitro transfection of human BARX2 cDNA induced cadherin 6 expression. Transfection of BARX2 into OAW42 inhibited Matrigel invasion, haptotactic cellular migration to a collagen IV signal, and adhesion to collagen IV-coated plates. Our data demonstrate that BARX2 is expressed in the ovarian surface epithelium and has functional suppressor properties in ovarian cancer cells.

Post-transcriptional silencing of RET occurs, but is not required, during raf-1 mediated differentiation of medullary thyroid carcinoma cells.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor of the calcitonin secreting thyroid C-cells. Somatic and germline mutations in the RET proto-oncogene are associated with sporadic and inherited cases of MTC, respectively. The human MTC cell line, TT, can be differentiated by activated raf-1. This differentiation is characterized, in part, by down-regulation of the RET proto-oncogene. We now show that raf-1 induction is followed by activation of the downstream kinases MEK1/2 and ERK1/2 and that differentiation is dependent on activation of MEK1/2. The concurrent down-regulation of RET appears to involve altered nuclear compartmentalization and transport of RET mRNA. Although RET is down-regulated during raf-1 mediated differentiation, overexpression of activated RET alleles which resist down-regulation does not alter the raf-1 mediated differentiation response. These data suggest that RET down-regulation is associated with, but not required, for raf-1 mediated MTC cell differentiation and that the raf-1 signal transduction pathway plays a dominant role in promoting MTC cell differentiation.