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Hepatocellular carcinoma (HCC) is the third leading cause of cancer death, and its incidence is increasing worldwide in an alarming manner. The development of curative therapy for advanced and metastatic HCC is a high clinical priority. The HCC genome is complex and heterogeneous; therefore, the identification of recurrent genomic and related gene alterations is critical for developing clinical applications for diagnosis, prognosis and targeted therapy of the disease. This article focuses on recent research progress and our contribution in identifying and deciphering the role of defined genetic alterations in the pathogenesis of HCC. A significant number of genes that promote or suppress HCC cell growth have been identified at the sites of genomic reorganization. Notwithstanding the accumulation of multiple genetic alterations, highly recurrent changes on a single chromosome can alter the expression of oncogenes and tumor suppressor genes (TSGs) whose deregulation may be sufficient to drive the progression of normal hepatocytes to malignancy. A distinct and highly recurrent pattern of genomic imbalances in HCC includes the loss of DNA copy number (associated with loss of heterozygosity) of TSG-containing chromosome 8p and gain of DNA copy number or regional amplification of protooncogenes on chromosome 8q. Even though 8p is relatively small, it carries an unusually large number of TSGs, while, on the other side, several oncogenes are dispersed along 8q. Compelling evidence demonstrates that DLC1, a potent TSG on 8p, and MYC oncogene on 8q play a critical role in the pathogenesis of human HCC. Direct evidence for their role in the genesis of HCC has been obtained in a mosaic mouse model. Knockdown of DLC1 helps MYC in the induction of hepatoblast transformation in vitro, and in the development of HCC in vivo. Therapeutic interventions, which would simultaneously target signaling pathways governing both DLC1 and MYC functions in hepatocarcinogenesis, could result in progress in the treatment of liver cancer.

The DLC1 gene encodes a Rho GTPase-activating protein (RhoGAP) that functions as a tumor suppressor in several common human cancers. The multidomain structure of DLC1 enables interaction with a number of other proteins. Here we report that the proinflammatory protein S100A10 (also known as p11), a key cell surface receptor for plasminogen which regulates pericellular proteolysis and tumor cell invasion, is a new binding partner of DLC1 in human cells. We determined that the 2 proteins colocalize in the cell cytoplasm and that their binding is mediated by central sequences in the central domain of DLC1 and the C-terminus of S100A10. Because the same S100A10 sequence also mediates binding to Annexin 2, we found that DLC1 competed with Annexin 2 for interaction with S100A10. DLC1 binding to S100A10 did not affect DLC1's RhoGAP activity, but it decreased the steady-state level of S100A10 expression in a dose-dependent manner by displacing it from Annexin 2 and making it accessible to ubiquitin-dependent degradation. This process attenuated plasminogen activation and resulted in inhibition of in vitro cell migration, invasion, colony formation, and anchorage-independent growth of aggressive lung cancer cells. These results suggest that a novel GAP-independent mechanism contributes to the tumor suppressive activity of DLC1, and highlight the importance and complexity of protein–protein interactions involving DLC1 in certain cancers. Cancer Res; 71(8); 2916–25. ©2011 AACR.

We have applied a functional gene transfer strategy to show the importance of viral integration site in cellular immortalization. The large tumor antigen of SV40 is capable of extending the cellular life span by sequestering tumor suppressor proteins pRB and p53 in virus-transformed human cells. Although SV40 large T antigen is essential, it is not sufficient for cellular immortalization, suggesting that additional alterations in cellular genes are required to attain infinite proliferation. We show here that the disruption of human chromosomal interval at 1q21.1 by SV40 integration can be an essential step for cellular immortalization. The transfer of a 150-kb bacterial artificial chromosome (BAC) clone, RP364B14, corresponding to viral integration site in CRL2504 cells, reverted their immortal phenotype. Interestingly, the BAC transfer clones of CRL2504 cells displayed characteristics of either senescence as shown by β-galactosidase activity or apoptosis as revealed by positive staining with M30 CytoDEATH antibody. The SV40 integration at 1q21.1, in the vicinity of epidermal differentiation complex (EDC) genes, resulted in the down-regulation of the filaggrin (FLG) gene that is part of the EDC. FLG gene expression was increased in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic sequence by SV40 may alter expression of genes involved in senescence and apoptosis by modulating chromatin structure. These studies imply that identification of genes located in the vicinity of viral integration sites in human cancers may be helpful in developing new diagnostic and therapeutic strategies. [Cancer Res 2009;69(19):7819–25]

Mouse models for hepatocellular carcinoma (HCC) provide an experimental ground to dissect the genetic and biological complexities of human liver cancer and enhance our ability to gain insights into the relevance of candidate cancer genes. We examined, using spectral karyotyping (SKY) and array-based CGH (aCGH), seven cell lines derived from HCC spontaneously developed in transgenic c-Myc animals (c-Myc), and four cell lines established from tumors induced in nude mice by inoculation with the original c-Myc cells (nuMyc). All of those cell lines exhibited gain of material from chromosomes 5, 6, 8, 10, 11, 15 and 19, and DNA copy-number loss from chromosomes 2, 4, 7, 9, 12, 14, and X. In addition, several recurrent chromosome reorganizations were found including del(3), t(3;8), del(4) t(4;11), t(6;5), del(7), del(8), del(9), t(10;14), del(11) and del(16). Chromosome breakpoints underlying rearrangements clustered in the regions previously identified as important for the early stages of c-Myc induced hepatocarcinogenesis. The results strongly suggest the importance of recurrent breakage and loss of chromosomes 4, 9 and 14, and gain of chromosomes 15 and 19 in mouse liver neoplasia. Genomic changes observed in c-Myc HCC cell lines are also recurrent in HCC developed in a different other transgenic mouse models, in mouse spontaneous HCC and derivative cell lines, as well as in preneoplastic liver lesions induced with chemical carcinogens. Overall, our results demonstrate selective, non-random genomic changes involving chromosomal regions homologous to those implicated in human HCC.

During the neoplastic process tumour cells frequently acquire resistance to the antiproliferative signals of transforming growth factor-beta (TGF-beta). Here we examined a human hepatocellular carcinoma cell line (Hep3B-TS) sensitive to TGF-beta signalling, and a derivative line (Hep3B-TR) rendered resistant to TGF-beta by stepwise exposure to TGF-beta(1). Comprehensive molecular cytogenetic analysis revealed that the acquisition of TGF-beta-resistance by Hep3B-TR cells was due to loss of TGF-beta receptor 2 (TGFbetaRII) gene. As demonstrated by spectral karyotyping and array-based comparative genomic hybridization, and in difference to Hep3B-TS cells, which have three rearranged and two normal copies of chromosome 3 that harbour the TGFbetaRII gene, Hep3B-TR cells have four rearranged and one apparently normal chromosome 3, which nonetheless underwent a critical microdeletion at the site of TGFbetaRII gene. Gene expression analysis using an oligonucleotide microarray of 21,397 genes showed that Hep3B-TR differentially expressed 307 genes, out of which 197 and 110 were up- and down-regulated, respectively, compared to Hep3B-TS. Six of differentially expressed genes were identified as downstream targets of the tumour necrosis factor (TNF) gene, suggesting that loss of TGFbetaRII triggered activation of the TNF pathway known to be regulated by TGF-beta(1) network. On the functional level, the TGF-beta-resistant Hep3B-TR cells displayed significantly enhanced capacity for anchorage independent growth and cell migration in vitro, and also increased tumorigenicity in vivo and in vitro and in vivo tumorigenicity compared with parental sensitive cells.

DLC1 (deleted in liver cancer 1), a tumor suppressor gene that encodes a RhoGTPase-activating protein, is recurrently downregulated or silenced in various solid tumors and hematological malignancies because of epigenetic modifications or genomic deletion. Here, we identified DLC1 promoter hypermethylation in 43 out of 44 multiple myeloma (MM) cell lines, which resulted in downregulation or silencing of DLC1 in 41 samples. High frequency of tumor-specific methylation and attenuation or silencing of DLC1 expression could serve as an independent diagnostic marker for MM. Combined treatment with demethylating and acetylating agents significantly elevated the expression of DLC1 and suppressed MM cell proliferation. Two cell lines exhibiting complete promoter methylation and the absence of DLC1 expression were transduced by an adenoviral vector containing DLC1 cDNA. In both cell lines, the reexpression of DLC1 inhibited myeloma cell invasion and migration, reduced RhoA activity and resulted in the reorganization of actin cytoskeleton. These results provide the first evidence for the antiproliferative effect of DLC1 in a hematological cancer and implicate RhoA pathway in suppression of MM migration and invasion. Given the myeloma cells sensitivity to the reactivation of DLC1 function, the potential for molecular targeted therapy of DLC1-mediated pathways as well as epigenetic therapies hold prospects.

The biological functions of nuclear topoisomerase I (Top1) have been difficult to study because knocking out TOP1 is lethal in metazoans. To reveal the functions of human Top1, we have generated stable Top1 small interfering RNA (siRNA) cell lines from colon and breast carcinomas (HCT116-siTop1 and MCF-7-siTop1, respectively). In those clones, Top1 is reduced ∼5-fold and Top2α compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone γ-H2AX foci associated with replication defects. siTop1 cells also show rDNA and nucleolar alterations and increased nuclear volume. Genome-wide transcription profiling revealed 55 genes with consistent changes in siTop1 cells. Among them, asparagine synthetase (ASNS) expression was reduced in siTop1 cells and in cells with transient Top1 down-regulation. Conversely, Top1 complementation increased ASNS, indicating a causal link between Top1 and ASNS expression. Correspondingly, pharmacologic profiling showed l-asparaginase hypersensitivity in the siTop1 cells. Resistance to camptothecin, indenoisoquinoline, aphidicolin, hydroxyurea, and staurosporine and hypersensitivity to etoposide and actinomycin D show that Top1, in addition to being the target of camptothecins, also regulates DNA replication, rDNA stability, and apoptosis. Overall, our studies show the pleiotropic nature of human Top1 activities. In addition to its classic DNA nicking-closing functions, Top1 plays critical nonclassic roles in genomic stability, gene-specific transcription, and response to various anticancer agents. The reported cell lines and approaches described in this article provide new tools to perform detailed functional analyses related to Top1 function. [Cancer Res 2007;67(18):8752–61]

To identify new antigens that are targets for the immunotherapy of prostate and breast cancer, we used expressed sequence tag and genomic databases and discovered POTE, a new primate-specific gene family. Each POTE gene encodes a protein that contains three domains, although the proteins vary greatly in size. The NH2-terminal domain is novel and has properties of an extracellular domain but does not contain a signal sequence. The second and third domains are rich in ankyrin repeats and spectrin-like helices, respectively. The protein encoded by POTE-21, the first family member discovered, is localized on the plasma membrane of the cell. In humans, 13 highly homologous paralogs are dispersed among eight chromosomes. The expression of POTE genes in normal tissues is restricted to prostate, ovary, testis, and placenta. A survey of several cancer samples showed that POTE was expressed in 6 of 6 prostate, 12 of 13 breast, 5 of 5 colon, 5 of 6 lung, and 4 of 5 ovarian cancers. To determine the relative expression of each POTE paralog in cancer and normal samples, we employed a PCR-based cloning and analysis method. We found that POTE-2α, POTE-2β, POTE-2γ, and POTE-22 are predominantly expressed in cancers whereas POTE expression in normal tissues is somewhat more diverse. Because POTE is primate specific and is expressed in testis and many cancers but only in a few normal tissues, we conclude POTE is a new primate-specific member of the cancer-testis antigen family. It is likely that POTE has a unique role in primate biology. (Cancer Res 2006; 66(1): 52-6)

We previously showed that the human AKNA gene encodes an AT-hook transcription factor that regulates the expression of costimulatory cell surface molecules on lymphocytes. However, AKNA cDNA probes hybridize with multiple transcripts, suggesting either the existence of other homologous genes or a complex regulation operating on a single gene. Here we report evidence for the latter, as we find that AKNA is encoded by a single gene that spans a 61-kb locus of 24 exons on the fragile FRA9E region of human chromosome 9q32. This gene gives rise to at least nine distinct transcripts, most of which are expressed in a tissue-specific manner in lymphoid organs. Many of the AKNA transcripts originate from alternative splicing; others appear to derive from differential polyadenylation and promoter usage. The alternative AKNA transcripts are predicted to encode overlapping protein isoforms, some of which (p70 and p100) are readily detectable using a polyclonal anti-AKNA antisera that we generated. We also find that AKNA PEST-dependent cleavage into p50 polypeptides is targeted to mature B cells and appears to be required for CD40 upregulation. The unusual capacity of the AKNA gene to generate multiple transcripts and proteins may reflect its functional diversity, and it may also provide a fail-safe mechanism that preserves AKNA expression.

We have identified an 85 kb BAC clone, 346J21, that carries a cell senescence gene (SEN16), previously mapped to 16q24.3. Transfer and retention of 346J21 in breast cancer cell lines leads to growth arrest after 8-10 cell doublings, accompanied by the appearance of characteristic senescent cell morphology and senescence-associated acid beta-galactosidase activity. Loss of transferred BAC results in reversion to the immortal growth phenotype of the parental cancer cell lines. BAC 346J21 restores senescence in the human breast cancer cell lines, MCF.7 and MDA-MB468, and the rat mammary tumor cell line LA7, but not in the human glioblastoma cell line T98G. We postulate that inactivation of both copies of SEN16 is required for the immortalization of breast epithelial cells at an early stage of tumorigenesis. Positional mapping of 346J21 shows that SEN16 is distinct from other candidate tumor suppressor genes reported at 16q24.

The WWOX (WW-domain containing oxidoreductase) is a candidate tumour suppressor gene spanning the same chromosome region, 16q23, as the second most common fragile site (FS), FRA16D. Deletions detected by comparative genomic hybridisation (CGH) and loss of heterozygosity at microsatellite markers on chromosome 16q are common in many human cancers including hepatocellular carcinoma (HCC). The development of human HCC is closely associated with exposure to oncogenic viruses and chemical carcinogens, agents known to frequently target common FS. We examined the status of WWOX genomic DNA, RNA and protein in 18 cell lines derived from human HCC and found recurrent alterations of the gene. Loss of DNA copy-number confined to band 16q23 was detected by CGH in several cell lines. Although homozygous deletions of the WWOX gene were not detected, WWOX mRNA expression was absent or lower in 60% of cell lines. The occurrence of aberrant WWOX reverse transcription-PCR products with deletion of exons 6-8 correlated significantly with altered WWOX expression. All of the cell lines showing mRNA downregulation had a decreased or undetectable level of WWOX protein as demonstrated by Western blotting with antibody to WWOX. Furthermore, 13 out of the 18 cell lines expressed decreased levels or no WWOX protein when compared with normal liver. These results show that WWOX gene is frequently altered in HCC and raise the possibility that this gene is implicated in hepatocarcinogenesis.

DNA topoisomerases contribute to various cellular activities that involve DNA. We previously identified a human nuclear gene that encodes a mitochondrial DNA topoisomerase. Here we show that genes for mitochondrial DNA topoisomerases (type IB) exist only in vertebrates. A 13-exon topoisomerase motif was identified as a characteristic of genes for both nuclear and mitochondrial type IB topoisomerases. The presence of this signature motif is thus an indicator of the coexistence of nuclear and mitochondrial type IB DNA topoisomerases. We hypothesize that the prototype topoisomerase IB with the 13-exon structure formed first, and then duplicated. One topoisomerase specialized for nuclear DNA and the other for mitochondrial DNA.

GC-binding factor 2 (GCF2) is a transcriptional repressor that decreases activity of the epidermal growth factor receptor (EGFR) and other genes. We have mapped the gene for GCF2 by fluorescence in situ hybridization (FISH) to chromosome 2q37. Sequence analysis of the GCF2 gene and cDNA showed that the gene consists of eight exons and introns and spans 73 kbp of DNA. Northern blot analysis showed that GCF2 mRNA was differentially expressed in many human tissues and cell lines. GCF2 mRNA was expressed as a 4.2 kb mRNA in most human tissues with the highest expression level in peripheral blood leukocytes and lowest expression in brain and testis. Additional transcripts of 6.6, 2.9 and 2.4 kb were found in some tissues but the only transcript detected in cancer cell lines was 4.2 kb with high levels found in seven Burkitts' lymphoma cell lines. Western blot analysis showed that GCF2 protein is present at high levels in Burkitts' lymphoma and several other cancer cell lines. GCF2 was found in both nuclear and cytoplasmic compartments in cells. Deletion mutants of GCF2 revealed that amino acids 429–528 are required for both DNA binding and repression of the EGFR promoter. Furthermore, GCF2 was able to substantially decrease activator protein 2 (AP2) enhancement of the EGFR promoter. Thus, GCF2 is a transcriptional repressor overexpressed in cancer cell lines with a role in regulating expression of the EGFR.

Transgenic mice expressing PML-RARα in early myeloid cells under control of human cathepsin G regulatory sequences all develop a myeloproliferative syndrome, but only 15% to 20% develop acute promyelocytic leukemia (APL) after a latent period of 6 to 14 months. However, this transgene is expressed at very low levels in the bone marrow cells of transgenic mice. Because the transgene includes only 6 kb of regulatory sequences from the human cathepsin G locus, we hypothesized that sequences required for high-level expression of the transgene might be located elsewhere in the cathepsin G locus and that a knock-in model might yield much higher expression levels and higher penetrance of disease. We, therefore, targeted a human PML-RARα cDNA to the 5′ untranslated region of the murine cathepsin G gene, using homologous recombination in embryonic stem cells. This model produced a high-penetrance APL phenotype, with more than 90% of knock-in mice developing APL between 6 and 16 months of age. The latent period and phenotype of APL (including a low frequency of an interstitial deletion of chromosome 2) was similar to that of the previous transgenic model. Remarkably, however, the expression level of PML-RARα in bone marrow cells or APL cells was less than 3% of that measured in the low-penetrance transgenic model. Although the explanation for this result is not yet clear, one hypothesis suggests that very low levels of PML-RARα expression in early myeloid cells may be optimal for the development of APL in mice.

Signaling by the transforming growth factor (TGF)-family members is transduced from the cell surface to the nucleus by the Smad group of intracellular proteins. Because we detected alterations on the long arm of chromosome 5, we examined the status of the SMAD5 gene in human hepatocellular carcinoma (HCC) cell lines and primary HCC. In 16 cell lines, chromosome alterations of chromosome 5 were observed in nine cell lines by fluorescence in situ hybridization (FISH), and an increase in SMAD5 gene copy number relative to the ploidy level was found in eight lines. The breakpoints in unbalanced translocations and deletions frequently occurred near the SMAD5 locus, but apparently did not cause loss of SMAD5. In one cell line, where comparative genomic hybridization showed DNA copy number gain confined to the region 5q31, we detected by FISH high-level amplification of the SMAD5 gene located within the fragile site FRA5C. Semiquantitative polymerase chain reaction did not reveal changes in SMAD5 DNA levels in 15 of 17 primary HCC specimens. In 17 HCC cell lines, SMAD5 mRNA levels were either maintained or upregulated by an increase in gene dosage or another mechanism. Collectively, our results show that SMAD5 undergoes copy number gain and increased expression, rather than loss of expression, and therefore suggest that this gene does not act as a tumor-suppressor gene in HCC. The Hep-40 HCC cell line with high-level amplification and significant overexpression of SMAD5 may be useful in studying the interaction of SMAD5 with other genes.

DNA amplification in cancer cells frequently involves oncogenes whose increased expression confers a selective advantage on tumor cell growth. In an attempt to identify novel oncogenes involved in hepatocarcinogenesis, representational difference analysis (RDA) was performed using DNA from a primary human hepatocellular carcinoma (HCC) that showed high-level DNA amplifications on chromosomes 1p32 and 11q13 by comparative genomic hybridization. Ten amplification fragments were isolated by RDA, and when used to probe Southern blots of tumor DNA, there was a 5- to 50-fold increase in hybridization intensity relative to normal DNA. The sequence of one amplification product matched that of the EMS1 oncogene, which is located on chromosome 11q13 and is amplified in other cancers. We detected EMS1 amplification in 3 of 17 primary HCC. Overexpression of EMS1 mRNA was observed in 12 of 14 HCC cell lines in the absence of gene amplification or an increased copy-number of the gene. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with tumor development in HCC. EMS1 amplification and overexpresion is indicative of unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer.

The human DLC-1 (deleted in liver cancer 1) gene was cloned from a primary human hepatocellular carcinoma (HCC) and mapped to the chromosome 8p21-22 region frequently deleted in common human cancers and suspected to harbor tumor suppressor genes. DLC-1 was found to be deleted or downregulated in a significant number of HCCs. We expanded our investigations to other cancers with recurrent deletions of 8p22, and in this study examined alterations of DLC-1 in primary human breast tumors, human breast, colon, and prostate tumor cell lines. Genomic deletion of DLC-1 was observed in 40% of primary breast tumors, whereas reduced or undetectable levels of DLC-1 mRNA were seen in 70% of breast, 70% of colon, and 50% of prostate tumor cell lines To see whether DLC-1 expression affects cell growth and tumorigenicity, two breast carcinoma cell lines lacking the expression of endogenous gene were transfected with the DLC-1 cDNA. In both cell lines, DLC-1 transfection caused significant growth inhibition and reduction of colony formation. Furthermore, introduction of the DLC-1 cDNA abolished the in vivo tumorigenicity in nude mice, suggesting that the DLC-1 gene plays a role in breast cancer by acting as a bona fide tumor suppressor gene.

Chromosomal abnormalities have been implicated in cancer development since the turn of the last century. Only during the past two decades, with advances in cytogenetics and molecular biology, has the genetic basis of neoplasia been firmly established, however, with chromosomal alterations being recognized as critical in the pathogenesis of human cancer. Recurrent chromosomal alterations provide cytological and molecular markers for the diagnosis and prognosis of disease. They also facilitate the identification of genes that are important in carcinogenesis and, ultimately, may lead to the development of targeted therapy. In breast cancer, the most prevalent malignancy among females, substantial progress has been achieved in identifying genes located at sites of recurrent chromosomal alterations and in profiling gene expression through the application of powerful cytogenetic and functional genomic techniques. Characterization of the molecular pathologic characteristics and gene-expression profiles of breast cancer should provide new clinical tools for the accurate diagnosis and prediction of prognosis as well as new targets for the development of therapeutic agents.

T/EBP/NKX2.1, also known as TTF-1, is a homeodomain-containing transcription factor that is expressed in lung, thyroid, and a part of the brain (15, 21, 22, 28). T/EBP/NKX2.1 was originally characterized as a transcription factor that regulates expression of thyroid-specific genes such as those encoding thyroglobulin (7), thyroid peroxidase (7, 11, 20), TSH receptor (6, 37), and the Na/I symporter (10). In the lung, T/EBP/NKX2.1 is expressed in all epithelial cells early during pulmonary morphogenesis, but the expression becomes progressively restricted to alveolar type II and Clara cells towards the end of gestation and in postnatal days (46). T/EBP/NKX2.1 activates transcription of genes specifically expressed in lung, including the genes for surfactant proteins A (2), B (1), and C (19) and Clara cell secretory protein (also called uteroglobin) genes (34, 35). Targeted disruption of the T/ebp/Nkx2.1 locus was shown to result in immediate postnatal death due to respiratory failure caused by profoundly hypoplastic lungs (21, 26). These mice also lack the thyroid, pituitary, and parts of the ventral forebrain such as the hypothalamus and basal ganglia (21, 41, 42). Detailed analyses of the T/ebp/Nkx2.1-null mouse respiratory system revealed that T/EBP/NKX2.1 may function in the establishment of pattern formation of early pulmonary structure and pulmonary morphogenesis during embryonic development (26). A role for T/EBP/NKX2.1 in pulmonary morphogenesis was also suggested by in vitro experiments in which a T/EBP/NKX2.1 antisense oligonucleotide inhibited normal branching morphogenesis in lung organ culture (25). Based on these results, it is hypothesized that lung branching morphogenesis must be related to the ability of T/EBP/NKX2.1 to activate and/or suppress specific downstream target genes. One such category of target genes in lung consists of the surfactant proteins A (2), B (1), and C (19) and Clara cell secretory protein genes (34, 35). However, they are not known to have morphoregulatory function. Extensive studies have shown that epithelial-mesenchymal interaction plays an instructive role in lung branching morphogenesis (3, 17, 24, 45). In this context, a possible function for T/EBP/NKX2.1 may be in activation of epithelial cell pathways that are necessary for receiving and/or interpreting the instructive signals that originate from the mesenchyme. In this role, downstream target genes for T/EBP/NKX2.1 would potentially include those encoding cell surface receptors, components of the signal transduction pathway, and/or a variety of other factors connecting the cell surface to changes in gene expression and cellular behavior. In T/ebp/Nkx2.1-null embryo lungs, expression of some extracellular matrix proteins and their cellular receptors, including collagen type IV and α integrins, and some growth factors such as VEGF3 and BMP4 is reduced or absent (46). Whether the abnormal phenotype in T/ebp/Nkx2.1-null embryo lungs is entirely or partially due to the reduction or absence of expression of these genes remains to be examined. Among possible target genes are those encoding elements of tight junctions (TJs), a specialized membrane domain at the most apical region of polarized epithelial cells that creates a primary barrier to prevent paracellular transport of solutes and restrict the lateral diffusion of membrane lipids and proteins to maintain cellular polarity (4, 5, 23, 27, 40, 44). Claudins, products of a recently identified multigene family, are components of TJ strands and have four transmembrane domains and two extracellular loops with both NH2 and COOH termini in the cytoplasm (29, 43). Claudins have several functional characteristics consistent with a role in barrier formation and dependent on the specific claudin species that exhibit tissue specificity (27, 29). Claudin-1 and -2 have the ability to induce the formation of networks of strands and grooves at cell-cell contact sites when introduced into fibroblasts lacking TJs (12). Both claudin-3 and -4 are receptors for a cytotoxic enterotoxin (CPE) produced by the bacterium Clostridium perfringens, and the interaction with CPE results in increased membrane permeability by forming small pores in plasma membrane (39). A claudin-11 (oligodendrocyte-specific protein) knockout mouse showed the absence of TJ strands in central nervous system myelin and Sertoli cells in testis (14). Mutations in human claudin-16 (paracellin-1) cause renal hypomagnesemia with hypercalciuria and nephrocalcinosis, suggesting that it creates a channel that allows magnesium to diffuse through renal TJs (38). To date, at least 20 members of the claudin gene family have been identified (27). However, many of these claudins have not yet been examined in detail, and the functional differences are largely unknown. Here, we isolated and characterized a novel member of claudin gene family, claudin-18, which has two isoform transcripts produced by alternative splicing that exhibit lung- and stomach-specific expression. Further, claudin-18 has a splicing variant lacking the C-terminal cytoplasmic domain. Analysis of the promoter function of the mouse claudin-18 gene suggests that the lung-specific form is a downstream target gene regulated by the T/EBP/NKX2.1 homeodomain transcription factor.

We previously generated a transgenic mouse model for acute promyelocytic leukemia (APL) by expressing the promyelocytic leukemia (PML)–retinoic acid receptor (RARα) cDNA in early myeloid cells. This fusion protein causes a myeloproliferative disease in 100% of animals, but only 15–20% of the animals develop acute leukemia after a long latency period (6–13 months). PML-RARα is therefore necessary, but not sufficient, for APL development. The coexpression of a reciprocal form of the fusion, RARα-PML, increased the likelihood of APL development (55–60%), but did not shorten latency. Together, these results suggested that additional genetic events are required for the development of APL. We therefore evaluated the splenic tumor cells from 18 transgenic mice with APL for evidence of secondary genetic events, by using spectral karyotyping analysis. Interstitial or terminal deletions of the distal region of one copy of chromosome 2 [del(2)] were found in 1/5 tumors expressing PML-RARα, but in 11/13 tumors expressing both PML-RARα and RARα-PML (P< 0.05). Leukemic cells that contained a deletion on chromosome 2 often contained additional chromosomal gains (especially of 15), chromosomal losses (especially of 11 or X/Y), or were tetraploid (P≤ 0.001). These changes did not commonly occur in nontransgenic littermates, nor in aged transgenic mice that did not develop APL. These results suggest that expression of RARα-PML increases the likelihood of chromosome 2 deletions in APL cells. Deletion 2 appears to predispose APL cells to further chromosomal instability, which may lead to the acquisition of additional changes that provide an advantage to the transformed cells.

The Ly-6 family includes a number of highly homologous, low molecular weight glycophosphatidylinositol-linked proteins expressed on hematopoietic and lymphoid cells. The best characterized family member is Sca-1 (Ly-6A/E), an antigen commonly used for purification of murine pluripotent hematopoietic cells. We sought to characterize the genomic locus surrounding the Sca-1 gene. We identified several overlapping P1 artificial chromosomes containing theSca-1 gene and mapped one of these to mouse chromosome 15D3.1-3.3, the region previously shown to contain members of the murine Ly-6 gene family. We then mapped this clone and found that the Sca-2 gene lies 35.4 kilobase (kb) downstream ofSca-1 in the opposite transcriptional orientation. This is the first direct demonstration of physical linkage of Ly-6 genes. A novel gene, highly homologous to Sca-1 was identified and localized 13.4 kb downstream of Sca-1. This gene, which we designated Ly-6M, shares several structural features conserved among members of the Ly-6 family. Ly-6M messenger RNA (mRNA) is easily detectable in hematopoietic tissue (bone marrow, spleen, thymus, peritoneal macrophages) as well as kidney and lung. No mRNA expression was detected in heart, stomach, liver, small intestine, brain, or skin. Ly-6M protein is detectable on 10% to 15% of peripheral blood leukocytes, including monocytes and a subpopulation of B220+ cells. Ly-6M is broadly distributed in the bone marrow, with prominent expression on monocytes and myeloid precursors. The identification and characterization of Ly-6M adds a new member to a complex family of homologous, tightly linked genes that have proven extremely useful reagents for defining populations within the hematopoietic system.

The RelA (p65) subunit of transcription factor NF-κB plays a critical role in development, and rela–/– knockout mice die in utero from massive liver apoptosis. Only partial sequences of the mouse Rela gene are available. We have determined the genomic structure of mouse Rela and promoter, and have mapped the gene to chromosome 19B1–3.

Mutations in the palmitoyl-protein thioesterase (PPT) gene cause infantile neuronal ceroid lipofuscinosis (INCL), the clinical manifestations of which include the early loss of vision followed by deterioration of brain functions. To gain insight into the temporal onset of these clinical manifestations, we isolated and characterized a murine PPT (mPPT)-cDNA, mapped the gene on distal chromosome 4, and studied its expression in the eye and in the brain during development. Our results show that both cDNA and protein sequences of the murine and human PPTs are virtually identical and that the mPPT expression in the retina and in the brain is temporally regulated during development. Furthermore, the retinal expression of mPPT occurs much earlier and at a higher level than in the brain at all developmental stages investigated. Since many retinal and brain proteins are highly palmitoylated and depalmitoylation by PPT is essential for their effective recycling in the lysosomes, our results raise the possibility that inactivating mutations of the PPT gene, as occur in INCL, are likely to cause cellular accumulation of lipid-modified proteins in the retina earlier than in the brain. Consequently, the loss of vision occurs before the deterioration of brain functions in this disease.

To search for recurrent and specific genomic alterations in human hepatocellular carcinoma (HCC), we examined 18 cell lines by comparative genomic hybridization (CGH), a molecular cytogenetic approach that allows positional identification of gains and losses of DNA sequences of the entire tumor genome. We report here a distinct pattern of multiple recurrent DNA copy-number gains and losses that include alterations frequently seen in other neoplasias as well as changes potentially specific for HCC. The most frequent gains were localized on 1p34.3-35, 1p33-34.1, 1q21-23, 1q31-32, 6p11-12, 7p21, 7q11.2, 8q24.1-24.2, 11q11-13, 12q11-13, 12q23, 17q11. 2-21, 17q23-24, and 20p11.1-q13.2. Recurrent losses were mapped on 3p12-14, 3q25, 4p12-14, 4q13-34, 5q21, 6q25-26, 8p11.2-23, 9p12-24, 11q23-24, 13q12-33, 14q12-13, 15q25-26, 18q11.2-22.2, and 21q21-22. Seventeen genomic imbalances are novel in HCC, thus extending significantly the map of genetic changes and providing a starting point for the isolation of new genes relevant in pathogenesis of liver neoplasia, as well as providing molecular probes for both diagnosis and monitoring treatment of the disease.

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9. 53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase alpha/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations.

The mammaglobin gene encodes a novel secreted protein whose corresponding mRNA is frequently up-regulated in human breast cancer. In non-malignant tissues, expression is also strictly limited to the mammary epithelium. To better understand the mechanisms controlling these patterns of expression, we have isolated the human mammaglobin gene and performed an initial assessment of its promoter activity. Mammaglobin gene architecture is very similar to that of a family of related genes that includes uteroglobin and rat prostatein subunits C1, C2, and C3. However, the mammaglobin gene itself is not well conserved phylogenetically. The human mammaglobin gene is localized by fluorescent in situ hybridization to chromosome 11 band q13, a genomic region frequently amplified in breast neoplasia. The sequence of proximal 1 kb of mammaglobin promoter contains several potential transcriptional control elements and directs high-level expression of a transfected reporter construct in human breast tumor cell lines. However, comparable levels of reporter gene expression are also seen in non-mammary human cell lines. These data suggest that, unlike related gene family members, the striking breast-specific expression and tumor-associated overexpression of mammaglobin is mediated by complex transcriptional control at more distal sequence elements.

DLC1 (Deleted in Liver Cancer 1) gene encodes a RhoGTPase-activating protein (RhoGAP), which exerts most of its tumor suppressor functions through suppression of small Rho GTPases proteins RhoA, RhoB, RhoC and to some degree Cdc42, but not Rac. RhoGTPases are implicated in NF-κB activation in highly invasive prostate carcinoma (PCA), with consequences on cell proliferation, survival and metastatic capacity. Here we demonstrate that DLC1 transduction in two androgen-independent (AI) and highly metastatic PCA cell lines negatively regulates NF-κB activity in a GAP- and α-catenin-dependent manner. Expressed DLC1 protein suppresses the phosphorylation of NF-κB inhibitor, IκBα, causes its relocation from membrane ruffles into cytoplasm and attenuates its ubiquitination and subsequent degradation. DLC1-mediated NF-kB suppression and its effects are comparable to NF-κB inhibition using either shRNA knockdown or peptide inhibitor. Expression of transduced DLC1 suppressed the expression of NF-κB mediated genes. Such effects were found to be reliant on presence of calcium, indicating that the observed modifications are dependent on, and enabled by DLC-mediated stabilization of adherens junctions. These results expand the multitude of DLC1 interactions with other genes that modulate its oncosuppressive function, and may have potential therapeutic implications.

Neurturin is a potent neurotrophic factor closely related to glial cell line-derived neurotrophic factor (GDNF, 40% amino acid sequence identity) and, like GDNF, can promote the survival of numerous neuronal populations including sympathetic, nodose, and dorsal root ganglion sensory neurons. Both neurturin and GDNF signal through the Ret tyrosine kinase and require a glycosylphosphatidylinositol-linked coreceptor. Mutations in Ret and GDNF cause intestinal aganglionosis and renal dysplasia. Activating Ret mutations also cause multiple endocrine neoplasia syndromes (MEN2A and MEN2B). We have isolated mouse and human genomic neurturin clones. The sequence for preproneurturin is encoded by two exons. Mouse and human clones have common intron/exon boundaries. We have used interspecific backcross analysis to localize neurturin to mouse chromosome 17 near the Vav locus and fluorescence in situ hybridization analysis to localize human neurturin to the syntenic region of human chromosome 19p13.3.

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.

Background Lung cancer is the leading cause of cancer-related mortality. Therapies against non-small cell lung cancer (NSCLC) are particularly needed, as this type of cancer is relatively insensitive to chemotherapy and radiation therapy. We recently identified GGTI compounds that are designed to block geranylgeranylation and membrane association of signaling proteins including the Rho family G-proteins. One of the GGTIs is P61A6 which inhibits proliferation of human cancer cells, causes cell cycle effects with G1 accumulation and exhibits tumor-suppressing effects with human pancreatic cancer xenografts. In this paper, we investigated effects of P61A6 on non-small cell lung cancer (NSCLC) cells in vitro and in vivo. Methods Three non-small cell lung cancer cell lines were used to test the ability of P61A6 to inhibit cell proliferation. Further characterization involved analyses of geranylgeranylation, membrane association and activation of RhoA, and anchorage-dependent and –independent growth, as well as cell cycle effects and examination of cell cycle regulators. We also generated stable cells expressing RhoA-F, which bypasses the geranylgeranylation requirement of wild type RhoA, and examined whether the proliferation inhibition by P61A6 is suppressed in these cells. Tumor xenografts of NSCLC cells growing in nude mice were also used to test P61A6’s tumor-suppressing ability. Results P61A6 was shown to inhibit proliferation of NSCLC lines H358, H23 and H1507. Detailed analysis of P61A6 effects on H358 cells showed that P61A6 inhibited geranylgeranylation, membrane association of RhoA and caused G1 accumulation associated with decreased cyclin D1/2. The effects of P61A6 to inhibit proliferation could mainly be ascribed to RhoA, as expression of the RhoA-F geranylgeranylation bypass mutant rendered the cells resistant to inhibition by P61A6. We also found that P61A6 treatment of H358 tumor xenografts growing in nude mice reduced their growth as well as the membrane association of RhoA in the tumors. Conclusion Thus, P61A6 inhibits proliferation of NSCLC cells and causes G1 accumulation associated with decreased cyclin D1/2. The result with the RhoA-F mutant suggests that the effect of P61A6 to inhibit proliferation is mainly through the inhibition of RhoA. P61A6 also shows efficacy to inhibit growth of xenograft tumor.

An in vitro multistage genital epithelial cell model for cervical cancer that parallels the in vivo neoplastic process has been developed using recombinant human papillomavirus (HPV) DNA and genital cells. HPV-16-immortalized genital cells are responsive to the genotoxic action of known chemical carcinogens (polycyclic hydrocarbons, alkylating agents or cigarette smoke condensate), but are not converted to malignancy. Ras oncogene and human herpes virus-2 did convert HPV immortalized cells to malignancy, whereas human herpes virus-6 infection only increased HPV expression. Human immunodeficiency virus did not infect genital cells.

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.

Using overlapping cosmids representing the vascular endothelial growth factor (VEGF) locus, the VEGF gene was mapped by fluorescence in situ hybridization to chromosome 6p12. This localization permits linkage analysis and the identification of gene interaction in the region, as well as alterations of the VEGF structure or expression in cancer cells with chromosome abnormalities.

Humans and animals undergo ageing, and although their primary cells undergo cellular senescence in culture, the relationship between these two processes is unclear. Here we show that gamma-H2AX foci (gamma-foci), which reveal DNA double-strand breaks (DSBs), accumulate in senescing human cell cultures and in ageing mice. They colocalize with DSB repair factors, but not significantly with telomeres. These cryptogenic gamma-foci remain after repair of radiation-induced gamma-foci, suggesting that they may represent DNA lesions with unrepairable DSBs. Thus, we conclude that accumulation of unrepairable DSBs may have a causal role in mammalian ageing.

The primary structure of human sorbitol dehydrogenase (SORD) was determined by cDNA and genomic cloning. The nucleotide sequence of the mRNA covers 2471 bp including an open reading frame that yields a protein of 356 amino acid residues. The gene structure of SORD spans approximately 30 kb divided into 9 exons and 8 introns. The gene was localized to chromosome 15q21.1 by in situ hybridization. Two transcription initiation sites were detected. Three Sp1 sites and a repetitive sequence (CAAA)5 were observed in the 5' noncoding region; no classical TATAA or CCAAT elements were found. The related alcohol dehydrogenases and zeta-crystallin have the same gene organization split by 8 introns, but no splice points coincide between SORD and these gene types. The deduced amino acid sequence of the SORD structure differs at a few positions from the directly determined protein sequence, suggesting allelic forms of the enzyme. High levels of SORD transcripts were observed in lens and kidney, as judged from Northern blot analysis.

E-cadherin is a cell-cell adhesion molecule that acts as a suppressor of cancer cell invasion and its expression is downregulated in many advanced, poorly differentiated, human cancers. In this study, we found that the expression of DLC1 (deleted in liver cancer 1) tumor-suppressor gene in metastatic prostate carcinoma (PCA) cells increased the expression of E-cadherin and resulted in an elevated rate of cell-cell aggregation as measured by aggregation assay. DLC1-mediated increase in E-cadherin expression was not dependent on α-catenin, a DLC1-binding protein associated with E-cadherin, and/or cellular density. The increase of E-cadherin expression occurred at mRNA level and relied on DLC1 RhoGAP function, leading to suppression of high level of RhoA-GTP and RhoC-GTP activity in metastatic PCA cells. Application of Rho/ROCK inhibitors produced the same effect as introduction of DLC1. Knocking down of RhoA produced a moderate increase in E-cadherin whereas knocking down of RhoC resulted in a significant increase of E-cadherin. Downregulation of E-cadherin caused by constitutively active RhoA(V14) and RhoC(V14) could not be reversed by expression of DLC1 in DLC1-negative cell line. DLC1-mediated suppression of metastatic PCA cells invasion was comparable with the one associated with ectopic E-cadherin expression, or caused by suppression of Rho pathway either by Rho/ROCK inhibitors, or by shRNA repression. This study demonstrates that DLC1 expression positively regulates E-cadherin and suppresses highly metastatic PCA cell invasion by modulating Rho pathway, which appears as a feasible therapeutic target in cancers with high activity of RhoGTPases.

The DLC1 (for deleted in liver cancer 1) tumor suppressor gene encodes a RhoGAP protein that inactivates Rho GTPases, which are implicated in regulation of the cytoskeleton and adherens junctions (AJs), a cell-cell adhesion protein complex associated with the actin cytoskeleton. Malignant transformation and tumor progression to metastasis are often associated with changes in cytoskeletal organization and cell-cell adhesion. Here we have established in human cells that the AJ-associated protein α-catenin is a new binding partner of DLC1. Their binding was mediated by the N-terminal amino acids 340 to 435 of DLC1 and the N-terminal amino acids 117 to 161 of α-catenin. These proteins colocalized in the cytosol and in the plasma membrane, where together they associated with E-cadherin and β-catenin, constitutive AJ proteins. Binding of DLC1 to α-catenin led to their accumulation at the plasma membrane and required DLC1 GAP activity. Knocking down α-catenin in DLC1-positive cells diminished DLC1 localization at the membrane. The DLC1-α-catenin complex reduced the Rho GTP level at the plasma membrane, increased E-cadherin's mobility, affected actin organization, and stabilized AJs. This process eventually contributed to a robust oncosuppressive effect of DLC1 in metastatic prostate carcinoma cells. Together, these results unravel a new mechanism through which DLC1 exerts its strong oncosuppressive function by positively influencing AJ stability.

The physiologic expression of the human multidrug resistance MDRI gene product P-glycoprotein is controlled in a tissue- and cell-specific manner, but the regulatory mechanisms have not been characterized in great detail. Studies by Kohno et al. [(1990) J Biol Chem 265:19690–19696] suggested that a tissue-specific enhancer element located approximately 10 kb upstream from the major MDRI transcription start site may act to increase the levels of transcription in cultured adrenal and kidney cells. Using this putative “MDR enhancer” as a probe, we isolated a 14 kb DNA fragment from a genomic DNA library prepared from human fetal liver. The restriction map and partial nucleotide sequence of this DNA fragment were consistent with the previously described data obtained for a similar piece of genomic DNA derived from human placenta by Kohno et al. (ibid.). Pulsed-field gel electrophoresis of large genomic DNA fragments, however, showed that the DNA sequences, including the putative “MDR enhancer,” were not linked to the MDRI gene. Fluorescence in situ hybridization analysis revealed that this enhancer-like element is located on chromosome 20 at band q13.1 and is, therefore, distinct from the MDR locus on chromosome 7, band q21.1. Thus, this putative regulatory element does not modulate the tissue specificity of expression of the MDRI gene in vivo, but may play a role in the regulation of expression of another, so far unknown gene. Genes Chromosom Cancer 10:267–274 (1994). © 1994 Wiley-Liss, Inc.

The preponderance of evidence indicates that a subset of human papillomaviruses are important etiological agents for cervical cancer. However, the necessity of other agents as well as cellular events is recognized because not all women with papillomaviruses develop cancer. Therefore, the exact role of papillomaviruses in the multistage carcinogenesis process is unclear. Regulation of specific viral genes is important to the malignant process. The current study demonstrates that human herpesvirus-6, another ubiquitous virus, can infect genital epithelial cells and upregulate the expression of relevant papillomavirus genes. Thus, it can be considered a cofactor for cancer.

The C4-1 cell line derived from a non-keratinizing squamous cell carcinoma of the uterine cervix contains integrated human papillomavirus-18 DNA. Fluorescence in situ hybridization of C4-1 cells demonstrated a single viral integration site at 8q22.1 on a derivative chromosome originating from an 8q; 12q translocation. 8q22 is a site of chromosome fragility and is also recombinogenic in several human malignancies. DNase I hypersensitivity of the integration site was studied with a cellular flanking probe. A hypersensitive site was detected within 3 kb from viral DNA. The integration sites are undermethylated in C4-1 and HeLa cells and fully methylated in tumor cell lines of other origin, such as lymphoid cells. Genes Chrom Cancer 9:28-32 (1994). © 1994 Wiley-Liss, Inc.

The process of cell dissemination from the primary tumors to distant sites is the most harmful event during cancer progression, and the leading cause of cancer death. We have previously demonstrated that restoration of DLC1 tumor suppressor gene expression in the DLC1-negative Focus and 7703K human hepatocellular carcinoma (HCC) cell lines induced caspase-3 mediated apoptosis, reduced cell growth in vitro and tumorigenicity in vivo and diminished the ability to migrate through Matrigel, a property suggestive of metastatic potential in vivo. We now show that subcutaneous tumors developing after inoculation of Focus and 7703K cells into nude mice disseminate cells to liver and lung, and this process is markedly suppressed by restoration of DLC1 expression. Inhibition of tumor cell dissemination was associated with lower levels of RhoA activity, an increase in rounded cells and a reduction in actin stress fibers and focal adhesion molecules that are of critical importance in cancer cell invasion and metastasis. In addition, DLC1 down-regulated the expression of osteopontin and matrix metalloproteinase-9, which are highly up-regulated in most primary HCC with associated metastases. These observations implicate the DLC1 gene in suppression of HCC cell dissemination and identify novel cellular and genetic alterations that contribute to prevention of metastasis, a life-threatening event in cancer progression.

A hallmark in the pathogenesis of cancer is the increased expression of heat shock proteins (Hsps) and other molecular chaperones observed in many tumor types, which is considered to be an adaptive response to enhance tumor cell survival. Heat shock transcription factor 1 (Hsf1) is a major transactivator of Hsp induction and has been proposed to affect tumor initiation and progression, regulating expression of Hsps and other molecular targets. In this report, we provide direct in vivo evidence that Hsf1 plays a critical role in the evolution of spontaneous tumors arising in p53(-/-) mice. Thus, loss of Hsf1 function did not prolong tumor-free survival, but surprisingly altered the spectrum of tumors that arose in p53(-/-) mice. Tumor development is rapid in p53(-/-) mice, which predominantly (about 70%) succumb to lymphomas. In contrast, hsf1(-/-)p53(-/-) mice rarely develop lymphomas (

Fgf8 (fibroblast growth factor 8) was initially cloned from a mouse mammary tumor cell line derived from the androgen-dependent Shionogi carcinoma 115. The androgen-inducible expression of Fgf8 in this tumor controls its androgen-dependent phenotype, thus stimulating interest in this gene as a possible factor in human prostate cancer and other androgen-sensitive cancers. However, apart from Shionogi carcinoma 115, the androgen inducibility of Fgf8 is controversial. In the present study, having not detected androgen-inducible expression of Fgf8 in other mouse mammary cell lines or mouse prostate, we examined the Shionogi carcinoma 115-derived S115 cell line for mouse mammary tumor virus (MMTV) insertions or other nearby DNA rearrangements that might explain the androgen inducibility of Fgf8 in these cells. Southern blotting did not detect MMTV insertions near Fgf8 but did reveal a specific DNA rearrangement 3.7 kb upstream of Fgf8 in S115 cells and in other cells (SC115) independently derived from Shionogi carcinoma 115. Spectral karyotyping of S115 cells and sequencing of the cloned rearrangement junctions indicate that Fgf8 is involved in a t(5;19) translocation. The chromosome 5 sequence joined to Fgf8 is immediately adjacent to Smr2 (submaxillary gland androgen-regulated protein 2) and includes Muc10 (mucin 10), two genes that we show are testosterone inducible in S115 cells, suggesting that the androgen-dependent expression of Fgf8 in Shionogi carcinoma 115 and derivative cells results from this translocation. Together, these results suggest that androgen inducibility is not an inherent property of the Fgf8 gene, which has implications regarding this gene's proposed role in the etiology of hormone-responsive cancers. Published 2005 Wiley-Liss, Inc.

The etiology and genetic alterations of follicular thyroid carcinoma are not well understood. By targeting a mutation (PV) into the thyroid hormone receptor beta gene (TRbetaPV mouse), we created a knock-in mutant TRbeta(PV/PV) mouse that spontaneously develop follicular thyroid carcinoma with progression to metastasis similar to human follicular thyroid carcinoma. This mouse model provides a valuable tool to ascertain the nature and the extent of genomic rearrangements that occur during carcinogenesis of the thyroid. Spectral karyotyping analysis (SKY) of seven cell lines derived from thyroid tumors developed in TRbeta(PV/PV) mice showed that all of them had abnormal karyotypes, with chromosome number ranging from near-diploid (39-42 chromosomes) to hypotetraploid (63-79 chromosomes). These seven cell lines also exhibited a variety of structural chromosomal aberrations, including common recurrent translocations and deletions. This SKY analysis shows that the development and progression of follicular thyroid carcinoma in knock-in TRbeta(PV/PV) mutant mice comprise recurrent structural and numerical genomic changes, some of which mimic those described in human thyroid cancer.

Contrast-enhanced, look-up-table (LUT)-inverted digital images of DAPI-banded chromosomes after fluorescence in situ hybridization (FISH) permit direct regional chromosomal localization of the fluorescent signals of single-copy gene probes. Improved quality and resolution of chromosome banding allowed a refined localization of the erb B-3 protooncogene from chromosome band 12813 to subbands 12q13.2-13.3. This procedure can be used for direct and precise mapping of single-copy genes on both normal and cancer-cell-rearranged chromosomes.

Mutations of the Tp53 tumor suppressor gene have been detected in 50% of all human cancers. The normal Tp53 gene product acts as a negative regulator of cell division or is associated with abnormal growth in certain differentiated cell types. The Tp53 gene was mapped by FISH to Syrian hamster chromosome 9 bands qb2.2→2.3 using a genomic DNA probe with a high degree of sequence identity to the human Tp53 gene.

The sphingolipid, ceramide, is a natural dietary constituent and a potent mediator of apoptosis. If left undegraded, it may induce apoptosis and cause disruption of cellular integrity. A potential mechanism to prevent ceramide-induced apoptosis in various organs may involve ceramidases that facilitate the degradation of ceramide. In this study, we first isolated and characterized the murine neutral ceramidase (N-CDase) gene, mapped its chromosomal location and determined its developmental and organ-specific expression. Then we used cultured mesangial cells as our in vitro model and mouse gastrointestinal (GI) tract as the in vivo model to determine the effects of an inhibitor of N-CDase, D-erythro-MAPP, to delineate whether N-CDase plays a role in preventing ceramide-induced apoptosis. Our results show that: (i) the structure of the murine neutral ceramidase gene is virtually identical to that of the human gene; (ii) it is localized on chromosome 19 at bands C2-C3 that is syntenic to human chromosome 10q24-26; (iii) N-CDase expression is developmentally regulated and it is expressed at high levels in cultured mesangial cells and in specific regions of the mouse small intestine; (iv) inhibition of N-CDase by D-erythro-MAPP leads to increased ceramide levels and consequent apoptosis in cultured mesangial cells; (v) mice treated with D-erythro-MAPP alone also caused apoptosis in the small intestine; and (vi) mice treated with D-erythro-MAPP prior to feeding C2 ceramide manifest markedly elevated levels of apoptosis in the GI tract raising the possibility that neutral ceramidase plays a detoxifying role against inadvertent stimulation of ceramide-induced apoptosis in organs that come in contact with this sphingolipid. We propose that N-CDase is an essential component of an innate detoxifying mechanism to prevent ceramide-induced apoptosis.

We have identified a gene located on chromosomes 21 that is expressed in normal and neoplastic prostate, and in normal testis, ovary, and placenta. We name this gene POTE (expressed in prostate, ovary, testis, and placenta). The POTE gene has 11 exons and 10 introns and spans ≈32 kb of chromosome 21q11.2 region. The 1.83-kb mRNA of POTE encodes a protein of 66 kDa. Ten paralogs of the gene have been found dispersed among eight different chromosomes (2, 8, 13, 14, 15, 18, 21, and 22) with preservation of ORFs and splice junctions. The synonymous:nonsynonymous ratio indicates that the genes were duplicated rather recently but are diverging at a rate faster than the average for other paralogous genes. In prostate and in testis, at least five different paralogs are expressed. In situ hybridization shows that POTE is expressed in basal and terminal cells of normal prostate epithelium. It is also expressed in some prostate cancers and in the LnCAP prostate cancer cell line. The POTE protein contains seven ankyrin repeats between amino acids 140 and 380. Expression of POTE in prostate cancer and its undetectable expression in normal essential tissues make POTE a candidate for the immunotherapy of prostate cancer. The existence of a large number of closely related but rapidly diverging members, their location on multiple chromosomes and their limited expression pattern suggest an important role for the POTE gene family in reproductive processes.

Once immortalized, human cells are susceptible to transformation by introduction of an oncogene such as ras . Several lines of evidence now suggest that the maintenance of telomere length is a major determinant of replicative lifespan in human cells and thus of the immortalized state. The majority of human tumor cells acquire immortality through expression of the catalytic subunit of telomerase (hTERT), whereas others activate an alternative mechanism of telomere maintenance (ALT) that does not depend on the actions of telomerase. We have examined whether ALT could substitute for telomerase in the processes of transformation in vitro and tumorigenesis in vivo . Expression of oncogenic H-Ras in the immortal ALT cell line GM847 did not result in their transformation. However, subsequent ectopic expression of hTERT in these cells imparted a tumorigenic phenotype. Indeed, this outcome was also observed after introduction of a mutant hTERT that retained catalytic activity but was incapable of maintaining telomere length. These studies indicate that hTERT confers an additional function that is required for tumorigenesis but does not depend on its ability to maintain telomeres.