Transformation of Bloom's syndrome fibroblasts by DNA transfection.

Nonmalignant diploid human fibroblast cells (GM3498B) derived from a skin biopsy of a patient with Bloom's syndrome have been transformed by transfection with DNA from a tumorigenic mouse cell line (Ha-8) carrying a single copy of the Harvey murine sarcoma virus (Ha-MuSV) genome. The transformed cell lines have an extended life-span, form colonies in agarose, and proliferate in nude mice--characteristics of neoplastic transformation. Like the parental cells, they also exhibit a high spontaneous level of sister chromatid exchanges. Finally, the transformed cells contain most, if not all, of the Ha-MuSV genome as well as the human rasH sequence. These experiments show that these diploid nonmalignant human cells can be used as recipients in transfection experiments for studying the genetic control of neoplastic transformation.

A novel human gene closely related to the abl proto-oncogene.

A DNA sequence related to the abl proto-oncogene was identified in human placenta. Molecular cloning and nucleotide sequence analysis revealed two putative exons whose predicted amino acid sequence was most homologous to the corresponding sequences of c-abl and v-abl but was related to other tyrosine kinase genes as well. The new sequence was localized by in situ hybridization and somatic cell genetic analysis to human chromosome 1q24-25, which differs from the location of any previously identified tyrosine kinase gene. The detection of a novel 12-kb transcript by this gene in human normal and tumor cells establishes it as a new member of the tyrosine kinase family that is closely related to but distinct from c-abl.

Deleted in liver cancer 3 (DLC-3), a novel Rho GTPase-activating protein, is downregulated in cancer and inhibits tumor cell growth.

Two related Rho GTPase-activating proteins, DLC-1 (deleted in liver cancer 1) and DLC-2, are emerging as bona fide tumor suppressor genes that inhibit cancer cell growth. In this report, we characterized a gene on chromosome Xq13 that encodes DLC-3 (also known as KIAA0189 and STARD8), a third member of the DLC family. The DLC-3 gene has transcripts with alternative 5' ends, one of which, DLC-3alpha, encodes an 1103-amino acid polypeptide highly similar to DLC-1 and DLC-2. A second isoform (DLC-3beta) would yield a protein lacking the N-terminal sterile alpha motif domain. The DLC-3 gene is widely expressed in normal tissues, but DLC-3 mRNA levels were low or absent in a significant number of breast, ovarian, liver and prostate cancer cell lines. Using a cancer profiling array to compare matched tumor and normal human tissues, downregulation of DLC-3 mRNA was observed in kidney, lung, ovarian, uterine and breast cancer samples. By quantitative reverse transcriptase-polymerase chain reaction, DLC-3 expression was reduced in primary prostate carcinomas relative to normal prostate tissue. Transfection of human breast and prostate cancer cells with a DLC-3alpha expression vector inhibited cell proliferation, colony formation and growth in soft agar. These results indicate that deregulation of DLC-3 may contribute to breast and prostate tumorigenesis.

TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

NAB2, a corepressor of NGFI-A (Egr-1) and Krox20, is induced by proliferative and differentiative stimuli.

Previous work had identified a corepressor, NAB1, which represses transcriptional activation mediated by NGFI-A (also known as Egr-1, zif268, and Krox24) and Krox20. These zinc finger transcription factors are encoded by immediate-early genes and have been implicated in a wide variety of proliferative and differentiative processes. We have isolated and characterized another corepressor, NAB2, which is highly related to NAB1 within two discrete domains. The first conserved domain of NAB2 mediates an interaction with the R1 domain of NGFI-A. NAB2 represses the activity of both NGFI-A and Krox20, and its expression is regulated by some of the same stimuli that induce NGFI-A expression, including serum stimulation of fibroblasts and nerve growth factor stimulation of PC12 cells. The human NAB2 gene has been localized to chromosome 12ql3.3-14.1, a region that is rearranged in several solid tumors, lipomas, uterine leiomyomata, and liposarcomas. Sequencing of the Caenorhabditis elegans genome has identified a gene that bears high homology to both NAB1 and NAB2, suggesting that NAB molecules fulfill an evolutionarily conserved role.

claudin-18, a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing.

T/EBP/NKX2.1, a member of the NKX family of homeodomain-containing transcription factors, regulates the expression of a number of genes in lung and thyroid. Here we describe the isolation and characterization of a novel target gene, termed claudin-18, that is down-regulated in the lungs of T/ebp/Nkx2.1-null mouse embryos. The gene product exhibits an amino acid sequence similar to those of the claudin multigene family of proteins that constitute tight junction strands in epithelial cells. The gene was localized by fluorescence in situ hybridization to mouse chromosome 9 at region 9E3-F1 and to human chromosome 3 at region 3q21-23. The claudin-18 gene has two promoters, each with its own unique exon 1 that is spliced to common exons 2 through 5. Alternative usage of these promoters leads to production of lung and stomach-specific transcripts. The downstream lung-specific promoter contains two T/EBP/NKX2.1 binding sites responsible for trans activation of the gene by T/EBP/NKX2.1 in lung cells. Only claudin-18 was down-regulated in T/ebp/Nkx2.1-null embryo lungs among 11 claudin transcripts examined. Furthermore, the claudin-18 transcript has an alternative 12-bp insertion derived from the 5' end of intron 4, which produces a C-terminally truncated isoform in lung and stomach. Immunohistochemistry demonstrated complete membrane localization of claudin-18 with small focal dots in the lung and stomach epithelial cells. Immunogold electron microscopy analysis revealed that claudin-18 is concentrated at the cell-cell borders of epithelial cells. These unique features suggest a potentially important role for claudin-18 in the structure and function of tight junctions in lung and stomach.

Nab1, a corepressor of NGFI-A (Egr-1), contains an active transcriptional repression domain.

Nab proteins constitute an evolutionarily conserved family of corepressors that specifically interact with and repress transcription mediated by three members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate-early gene transcription factors, which includes NGFI-C, Krox20, and Egr3. We explored the mechanism of Nab1 repression and identified structural domains required for Nab1 function. Nab1 does not act by blocking DNA binding or nuclear localization of NGFI-A. In fact, Nab1 repression is not unique to NGFI-A because multiple types of non-NGFI-A activation domains were repressed, as was a heterologous transcription factor carrying the NGFI-A R1 domain, which is required for Nab1 interaction. Additionally, Nab1 tethered directly to DNA repressed constitutively active promoters. Tethered repression was not dependent on the identity of the basal promoter elements, the presence of a distal enhancer, or the distance separating the binding sites from the promoter. These results suggest that Nab1 repression is not specific to particular activators and that Nab1 is an active repressor that works by a direct mechanism. We identified a bipartite-like nuclear localization sequence and localized the repression function to the Nab conserved domain 2 (NCD2), a region found in the carboxy-terminal half of all Nab proteins. Three small regions of homology between Nab1 and previously characterized corepressors, Dr1 and E1b 55-kDa protein, were identified within NCD2. Replacement mutagenesis of residues conserved between these proteins interfered with Nab1 repression, although Nab1 does not function by the same mechanism as Dr1. The human NAB1 genomic locus was mapped to chromosome 2q32.3-33.

Sister chromatid exchange and chromosome aberration analysis with the use of several carcinogens and noncarcinogens.

Several chemical carcinogens and noncarcinogens were tested for their ability to induce sister chromatid exchanges (SCE) and structural chromosome aberrations in cultured V79-4 Chinese hamster cells. All of the direct-acting carcinogens induced a large increase in SCE frequency. Two chemicals, which are mutagenic in microorganisms but whose carcinogenicity is poorly documented, also increased the frequency of SCE. Carcinogenic polycyclic hydrocarbons caused an increased incidence of SCE only when a metabolizing feeder layer was used, whereas no increase was observed with noncarcinogenic polycyclic hydrocarbons. The other noncarcinogens also did not influence the SCE frequency. Although some chemicals increased the frequency of structural chromosome aberrations, no correlation was found between the frequencies of SCE and aberrations.

Deletion and translocation involving chromosome 3 (p14) in two tumorigenic Kaposi's sarcoma cell lines.

BACKGROUND: Two neoplastic Kaposi's sarcoma (KS) cell lines, KS Y-1 (derived from a patient with KS associated with acquired immunodeficiency syndrome) and KS SLK (derived from an immunosuppressed patient with a renal transplant and KS or iatrogenic KS), have been shown to have abnormal chromosome constitution and to require no exogenous growth factors. They produce malignant tumors in immunodeficient mice. In contrast, all other cell cultures prepared in the past from KS specimens have shown to have normal diploid characteristics are hyperplastic, and depends on cytokines for growth, but they do not produce malignant tumors in immunodeficient mice. PURPOSE: We investigated whether the chromosomal changes that occurred in these KS cell lines were random contribute to the pathogenesis of KS. METHODS: We used the conventional G-banding technique and fluorescence in sti hybridization to identify structural and numerical chromosomal changes in the KS cell lines. RESULTS: We demonstrated that both cell lines are aneuploid and have some additional features in common, i.e., loss of copies of chromosomes 14 and 21 and nonrandom translocations and deletions in the short arm of chromosome 3 at region 3p14. These KS cell lines also exhibits loss of heterozygosity of loci at region 3p14-ter. CONCLUSION: This is the first time nonrandom chromosomal alterations have been described in KS neoplastic cells. On the basis of information available on other available on other cancers, the chromosome 3 alterations observed here can be expected to contribute to the neoplastic process in KS. IMPLICATIONS: Future research should focus on the identification cytogenetic markers, thus facilitating generation of specific molecular probes for detecting neoplastic cells early in the disease process.

Chromosome alterations in Syrian hamster cells transformed in vitro by sodium bisulfite, a nonclastogenic carcinogen.

Sodium bisulfite, a nonmutagen at neutral pH, induces neoplastic transformation of cultured Syrian hamster fetal cells. Morphologically transformed fibroblast colonies were isolated, and derived cell lines formed anchorage-independent colonies in agarose and progressively growing s.c. fibrosarcomas in nu/nu mice. Five tumorigenic cell lines analyzed by G- and C-banding were chromosomally abnormal with numerical deviations and structural alterations. Three tumors that developed in nude mice had the chromosome constitution of the inoculated transformed cell as well as secondary changes associated with tumor progression. Transformed cell lines had either a predominantly near-diploid or a near-tetraploid population with consistent chromosome gain and loss. Monosomy of the chromosome 13 observed in three cell lines was a nonrandom numerical alteration. Four lines had abnormal chromosomes resulting from deletions, unbalanced translocations, or centric fusions, and one cell line had a chromosome with a homogeneously staining region. Changes of chromosomes 1 and X were observed in three lines. The breakpoints on X chromosome nonrandomly involved the region qa5 which is frequently affected in hamster cells transformed by other carcinogens and may result in loss of genes essential for the maintenance of a normal phenotype. The formation of abnormal chromosomes cannot be directly attributed to the initial DNA damage as bisulfite concentrations effective in causing neoplastic transformation induced a significant but minimal increase in sister chromatid exchanges and failed to cause chromosome aberrations. Bisulfite inhibition of DNA replication might be a contributing factor in the occurrence of abnormal chromosomes. This cytogenetic analysis provides the first evidence that neoplastically transformed cells by a nonclastogenic carcinogen exhibit persistent chromosome rearrangements, a genetic alteration essential to the process of malignant transformation.

Integration of human papillomavirus 16 DNA and genomic rearrangements in immortalized human keratinocyte lines.

Five foreskin-derived keratinocyte lines, immortalized by transfection of human papillomavirus (HPV16) DNA, were cytogenetically abnormal, exhibiting numerical deviations and altered chromosomes due to translocations, deletions, achromatic lesions, or partial duplications. Furthermore, all lines had cells with either homogeneously staining regions or double minute chromosomes, alterations associated with malignancy or drug resistance. None of these lines were tumorigenic in nude mice, showing that such alterations which are a manifestation of DNA amplification also occur in nonneoplastic cells. By in situ chromosome hybridization, viral sequences were identified on abnormal chromosomes at the junction of chromosome translocations, at achromatic lesions and within homogeneously staining regions and duplicated chromosome segments. Thus, for the first time in an experimental system, HPV16 integration into the cellular genome was associated with the induction of a subset of chromosome alterations. HPV16 integration that frequently occurred at fragile sites and near protooncogenes may be a critical alteration which confers a selective growth advantage and an indefinite proliferative potential to HPV-transfected cells.

Nonrandom chromosome alterations in human malignant mesothelioma.

Malignant mesothelioma (MM) is a neoplasm closely associated with asbestos exposure, which has been implicated in 70-80% of the cases. In this study, nine MM (two fresh surgical specimens, two permanent cell lines, and five xenografts in nude mice) were examined cytogenetically. Six patients had a known history of asbestos exposure. Seven MM were chromosomally abnormal, the majority having complex structural alterations affecting different chromosomes, whereas two fresh surgical specimens had a normal chromosome constitution. Alterations of chromosome 3 were detected in seven cases and changes involving chromosomes 1 and 7 were observed in six cases. The breakpoints of translocations and deletions on chromosome 1 involved several bands; however, 50% of the breakpoints were near the locations of Blym, L-myc, and ski protooncogenes. Forty % of the breaks on chromosome 7 involved bands q11.1-11.2 and 20% were at q22, the location of the met protooncogene. Nonrandom changes on chromosome 3 were interstitial or terminal deletions, and translocations involving the region p14-21. The deleted 3p segment was identifiable as part of a chromosome translocation in one MM and was apparently lost in the other six. The deletions involving 3p are either spontaneous or asbestos-induced lesions at vulnerable genomic sites and are the most common and nonrandom chromosome alterations observed. Possibly 3p abnormalities are causally related to the development of this malignancy.

Amplification of the integrated viral transforming genes of human papillomavirus 18 and its 5'-flanking cellular sequence located near the myc protooncogene in HeLa cells.

The human papillomavirus type 18 integrated in the HeLa cell genome is amplified on chromosome 8. E6, E7, and E1 open reading frames are amplified 5-fold, and the late viral DNA region, the viral long control region, and cellular flanking sequences are amplified 15-fold. The common 5'-flanking cellular DNA was localized by in situ hybridization of normal and HeLa cells only on chromosome 8 band q24. This flanking probe is included in the amplification unit of Colo320 cells, but in the case of HeLa the amplified region does not include the myc gene which is structurally conserved. Viral integration on chromosome 8 represents an independent event and not a rearrangement of viral DNA located on other chromosomes. The amplification of HPV-18 E6 and E7 open reading frames and the constitutive expression of the myc protooncogene may contribute to immortalization and/or proliferative capacity of HeLa cells.

Correlation of morphological transformation to sister chromatid exchanges induced by split doses of chemical or physical carcinogens on cultured Syrian hamster cells.

The relationship between the induction of DNA damage as reflected by sister chromatid exchange (SCE) formation and morphological transformation in exponentially growing Syrian hamster embryo cells was determined quantitatively after split doses of chemical or physical carcinogens. With split doses of carcinogen separated by 2 to 24 hr, only N-acetoxy-2-fluorenyl-acetamide (0.50 microgram/ml) enhanced both SCE induction and transformation when compared to single exposure. Split doses of N-methyl-N'-nitro-N- nitrosoguanidine (0.20 microgram/ml), mitomycin C (50 ng/ml), or ultraviolet light (3.0 J/sq m) were less effective than single exposures, while split doses of methyl methanesulfonate (40 micrograms/ml) caused transformation frequencies similar to a single treatment and decreased SCE frequencies with time intervals greater than 4 hr. Split or single exposures of X-irradiation (200 R) resulted in similar low frequencies of transformation and SCE. Contrasting with these results, a significant potentiation of SCE occurred after split doses of N-methyl-N'-nitro-N-nitrosoguanidine in cultures arrested in G1 with arginine-glutamine-deficient medium or by contact inhibition compared to a single treatment. This response was attributed to the interaction of carcinogen with DNA containing unrepaired damage and demonstrates the importance of the cell cycle phase of the target cell during carcinogen exposure for the induction of SCE by split doses of N-methyl-N'-nitro-N-nitrosoguanidine. The similarity of responses for transformation and SCE induction with split doses of carcinogens suggests that DNA lesions involved in SCE are essential for the initiation of neoplastic development.

Chromosome patterns (G and C bands) of in vitro chemical carcinogen-transformed guinea pig cells.

The chromosomes of five chemical carcinogen-transformed strain 2 guinea pig fetal cell lines were identified by G and C banding techniques and were compared with the normal karyotype from secondary untreated cultures. One line transformed by benzo(a)pyrene had a diploid constitution with no G and C band alterations. Three lines were near diploid, one was near tetraploid, and each contained abnormal chromosomes. A 3-methylcholanthrene line had an abnormal metacentric chromosome formed by centric fusion of two nonhomologous autosomes. The three N-methyl-N'-nitrosoguanidine or diethylnitrosamine cell lines exhibited submetacentric or subtelocentric abnormal chromosomes originating from translocations between two No. 1 or a No. 1 and another autosome. The involvement of Chromosome 1 may be due to its association with nucleolar organization. The greater frequency of contact between such chromosomes, compared to other autosomes, creates an increased risk of chromatid exchange possibly explaining their frequent participation in abnormal chromosome formation or nondisjunction.

Chromosome alterations associated with in vitro exposure of human fibroblasts to chemical or physical carcinogens.

Normal human foreskin fibroblasts treated in vitro with a chemical carcinogen or irradiated with ultraviolet light subsequently acquired anchorage independent growth and an extended but finite capacity for exponential growth. All cell lines were derived from cells recovered from colonies that had grown in semisolid medium; cell lines originally treated with a chemical carcinogen produced nodules after s.c. inoculation into nude mice. G-banding analysis of 10 cell lines (including one ultraviolet light line) revealed that seven were chromosomally abnormal with structural and numerical chromosome alterations, one was characterized by a consistent trisomy, and the other two were normal diploid. Structural alterations consisted of chromosome deletions, translocations, and partial chromosome duplications. Although no common structural or numerical abnormality was detected, several structural alterations were observed involving chromosomes 1, 7, 11, and 22, where fgr, erb-B, H-ras-1, and sis protooncogenes, respectively, are located. In one cell line trisomy 17 was a unique chromosome alteration. The induction of chromosome changes may have influenced the proliferative capacity of the treated cells relative to nontreated cells. However, the two cell lines without detectable chromosome changes also had an increased proliferative life span, suggesting that other submicroscopic genetic alterations may have affected cell multiplication. Although carcinogen induced chromosome abnormalities may represent a step in the process of neoplastic development, additional genetic and/or epigenetic changes, are required for indefinite growth and the expression of malignancy.

Expression and chromosomal localization of the cytochrome P1-450 gene in human mitogen-stimulated lymphocytes.

Genetic differences in aryl hydrocarbon hydroxylase (AHH) (flavoprotein-linked monoxygenase EC 1.14.14.1) activity in cultured lymphocytes have been linked with individual risk for certain environmentally caused cancers. Cytochrome P1-450 is the form of cytochrome P-450 most closely associated with AHH activity. In this study the chromosomal localization and the expression of human cytochrome P1-450 gene were determined in phytohemagglutinin-stimulated lymphocytes. In situ hybridization analysis provides assignment of the structural gene for human cytochrome P1-450 to chromosome 15q22-q24. Treatment of lymphocytes with benzanthracene increased the amount of mRNA hybridized to the cloned cytochrome P1-450 gene. The level of cytochrome P1-450 mRNA in these lymphocytes correlates well with the induced AHH activity indicating that non-cytochrome P1-450 enzymes contribute little to the individual differences in the level of AHH activity in the lymphocytes. Southern analyses of genomic DNA from individuals with high and low induced AHH activity demonstrated no detectable differences in the pattern or intensity of restriction fragments after treatment with benzanthracene from either individual. This finding together with the excellent correlation between the induced cytochrome P1-450 and AHH activity, suggests that transcriptional control rather than gene amplification or gross form of gene rearrangement accounts for cytochrome P1-450 induction in man. Measurements of cytochrome P1-450 mRNA content in cultured lymphocytes provide an alternative approach to the assay of AHH activity in assessing AHH phenotype and predicting different susceptibilities to deleterious environmental agents.

Alterations of the FHIT gene in human hepatocellular carcinoma.

FHIT (fragile histidine triad), a candidate tumor suppressor gene, encompasses FRA3B, a region with the highest fragility in the human genome, and is altered in a large number of human cancers, particularly those of epithelial cell origin and associated with known carcinogenic agents. Human hepatocellular carcinoma (HCC), a major cancer worldwide, is closely related to carcinogenic agents such as hepatitis B and C virus infections, dietary aflatoxin, alcohol consumption, and exposure to chemical carcinogens. To assess the extent and the nature of the FHIT gene alterations and their implications in the development of HCC, several cell lines and primary tumors were cytologically and molecularly examined. The FHIT gene is expressed in normal hepatic cells and is not expressed or is abnormally expressed in cultured tumor cells derived from HCC. Down-regulation of the FHIT gene was detected by Northern blot analysis in 9 of 14 cell lines However, neither abnormal FHIT transcripts nor point mutations in DNA sequences of reverse transcription-PCR products (exons 2-9) were identified. Expression of FHIT protein was not detected by immunostaining in 5 of 10 primary tumors. Four cell lines showing mRNA down-regulation did not express FHIT protein as demonstrated by Western blot analysis. Allelic loss of intron 5 of the FHIT gene was detected in 10 of 34 informative samples from primary tumors. Structural alterations of chromosome 3p were identified in 8 of 13 HCC cell lines. Deletions or translocations involving region 3p14.2 were identified by fluorescence in situ hybridization with a YAC850A6 probe spanning the FHIT locus on chromosomes derived from cell lines with an abnormal FHIT gene expression. These combined results indicate that the FHIT gene is a frequent target and may be implicated in a subset of liver cancers.

Positions of chromosome 3p14.2 fragile sites (FRA3B) within the FHIT gene.

The FHIT gene spans approximately 1 Mb of DNA at chromosome band 3p14.2, which includes the familial renal cell carcinoma chromosome translocation breakpoint (between FHIT exons 3 and 4), the most frequently expressed human constitutive chromosomal fragile site (FRA3B, telomeric to the t(3;8) translocation), and numerous homozygous deletions in various human cancers, frequently involving FHIT exon 5. The FRA3B has previously been shown to represent more than one specific site, and some specific representatives of FRA3B breaks have been shown to fall in two regions, which we know to be in FHIT introns 4 and intron 5. Because breakage and integration of exogenous DNA in this chromosome region is frequent in aphidicolin-treated somatic cell hybrids, cancer cells, and, presumably, aphidicolin-treated normal lymphocytes that exhibit gaps or breaks, we determined by one- and two color fluorescence in situ hybridization, using cosmids covering specific regions of the FHIT gene, that most of the aphidicolin-induced gaps at FRA3B fall within the FHIT gene, with the highest frequency of gaps falling in intron 5 of the FHIT gene, less than 30 kb telomeric to FHIT exon 5. Gaps also occur in intron 4, where a human papillomavirus 16 integration site has been localized, and in intron 3, where the t(3;8) break point is located. These results suggest that the cancer-specific deletions, which frequently involve introns 4 and 5, originated through breaks in fragile sites.

Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP.

The isolation of genes involved in cancer development is critical for uncovering the molecular basis of cancer. We report here the isolation of the full-length cDNA and chromosomal localization of a new gene frequently deleted in liver cancer (DLC-1) that was identified by representational difference analysis. Loss of heterozygosity was detected for DLC-1 in 7 of 16 primary hepatocellular carcinomas (HCCs) and in 10 of 11 HCC cell lines. Although mRNA for DLC-1 was expressed in all normal human tissues, it was not expressed in 4 of 14 HCC cell lines. Full-length cDNA for DLC-1 of 3800 bp encodes a protein of 1091 amino acids, has 86% homology with rat p122 RhoGAP gene, and was localized by fluorescence in situ hybridization on chromosome 8 at bands p21.3-22. Deletions on the short arm of chromosome 8 are recurrent in liver, breast, lung, and prostate cancers, suggesting the presence of tumor suppressor genes. DLC-1 may be a tumor suppressor gene in liver cancer as well as in other cancers.