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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Ninjurin is a novel protein that is up-regulated after nerve injury both in dorsal root ganglion (DRG) neurons and in Schwann cells. We previously reported that ninjurin demonstrates properties of a homophilic adhesion molecule and promotes neurite outgrowth from primary cultured DRG neurons. We have now found that ninjurin is widely expressed in both adult and embryonic tissues, primarily in those of epithelial origin. Aggregation assays were used to demonstrate that ninjurin-mediated adhesion requires divalent cations and is an energy-dependent process. The critical domain for ninjurin-mediated homophilic adhesion was localized to an 11-residue region (between Pro26 and Asn37) by mutagenesis and by employing synthetic oligopeptides as competitive inhibitors of ninjurin-mediated adhesion. Of particular importance are the Trp residue at position 29 and the 3 arginines in the region. Furthermore, we show that the peptide which inhibits aggregation of Jurkat cells expressing ninjurin is also capable of blocking the ability of ninjurin to promote neurite extension from DRG neurons. Using FISH analysis, the ninjurin gene was localized to human chromosome 9q22. Several genetic diseases of unknown etiology have been mapped to this region, including hereditary sensory neuropathy type 1, self-healing squamous epithelioma, split-hand/foot deformity type 1, and familial dilated cardiomyopathy.

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.

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.

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.

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 (

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.

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.

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.

In various studies of sporadic breast cancers, 40-70% were strongly positive for fragile histidine triad (Fhit) protein expression, whereas only 18% of BRCA2 mutant breast cancers demonstrated strong Fhit expression, suggesting that the BRCA2 repair function may be necessary to retain intact fragile common chromosome fragile site 3B(FRA3B)/FHITloci. In the current study, 22 breast tumors with deleterious BRCA1 mutations were analyzed for Fhit expression by immunohistochemistry in a case-control matched pair analysis. Loss of Fhit expression was significantly more frequent in the BRCA1 cancers compared with sporadic breast tumors (9% Fhit positive versus 68% Fhit positive), suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To investigate the relationship between repair gene deficiencies and induction of chromosome fragile sites in vitro, we have analyzed the frequency of aphidicolin induction of chromosome gaps and breaks in PMS2-, BRCA1-, MSH2-, MLH1-, FHIT-, and TP53-deficient cell lines. Each of the repair-deficient cell lines showed elevated expression of chromosome gaps and breaks, consistent with the proposal that proteins involved in mismatch and double-strand break repair are important in maintaining the integrity of common fragile regions. Correspondingly, genes at common fragile sites may sustain elevated levels of DNA damage in cells with deficient DNA repair proteins such as those mutated in several familial cancer syndromes.

In this study, CA46 and ST486, two Epstein-Barr (EBV) negative cell lines derived from sporadic BL, were analyzed by multicolor spectral karyotyping, G-banding, fluorescence in situ hybridization with single-copy gene probes, and comparative genomic hybridization (CGH). In addition to reciprocal t(8;14)(q24;q32) translocation involving c-myc and IgH loci, we identified a t(7;8;14)(q11.2;q24;q32) translocation in CA 46 cells and t(8;14;18)(q24;q32;q23) in ST486 cells. Both rearrangements were not previously described in BL and resulted in transposition of myc sequences in a new genomic configuration. Several DNA imbalances mapped by CGH at the same sites in both lines, may reflect recurrent genomic changes that are relevant to pathogenesis of BL. We tested the tumorigenicity of these lines by injecting cells intraperitoneally in SCID mice. In two separate experiments, CA46 cells produced tumors 2 weeks after cell inoculation while ST486 cells induced only one tumor after a long latency period. Partial duplication of the long arm of chromosome 1 involving variable bands but always band 1q23 is the second most common alteration in BL and is known to be associated with aggressive tumors and poor prognosis. Duplication of the bands 1q23-24 commonly observed in EBV-negative lines was identified only in highly tumorigenic CA46 cells suggesting that this region harbor gene(s) associated with tumor cell invasiveness.

International audience; While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

DNA amplification is associated with genomic instability, the main characteristic of cancer cells, and it frequently involves protooncogenes. Double minute chromosomes (DM) and homogeneously stained regions (HSR) are cytological manifestations of DNA amplification. Gain of chromosome 19 is a recurrent alteration in mouse hepatocellular carcinoma (HCC). In one tumor cell line established from HCC developed in myc transgenic mice, DM derived from chromosome 19 were identified by spectral karyotyping and confirmed by fluorescence in situ hybridization (FISH). A probe generated by PCR from microdissected DM was localized by FISH on normal and HCC-derived cell lines on DM and chromosome 19 at two sites separated by several medium size G-bands. This organization of DM containing amplified sequences from separate loci of the same chromosome, indicates a complex mechanism of DNA amplification, possibly involving more than one gene. DM or HSR were not previously identified in mouse HCC and adult human HCC. The recognition of these loci could lead to the cloning of new genes or identification of known genes important in development or progression of HCC.

Farber disease is an autosomal recessive disorder caused by lysosomal acid ceramidase (AC) deficiency. It commonly manifests during the first few months after birth with a unique triad of painful and progressive deformed joints, subcutaneous nodules, and progressive hoarseness. In order to understand the molecular mechanism(s) of pathogenesis of Farber disease, we isolated and characterized a full-length human AC gene, mapped its chromosomal location, determined the tissue-specific expression, and analyzed mutations in Farber disease patients. We also studied the AC-mRNA expression in gastrointestinal tumors and adjoining normal tissues. In addition, we determined the pattern of tissue-specific AC-mRNA expression in the adult mouse and during fetal development. Our results show that human AC gene consists of 14 exons and 13 introns spanning approximately 26.5 kb of genomic DNA. It is mapped to human chromosome 8p22–21.2, a region often disrupted in several cancers. The AC-mRNA is expressed in the mouse fetus from the seventh day of gestation. Interestingly, while the AC-mRNA is expressed in all segments of the normal gastrointestinal tract, none of the gastrointestinal tumor tissues had any AC-mRNA expression. We also uncovered four novel mutations in Farber disease patients that were not previously reported. Taken together, our results not only attest to the physiological importance of AC but also uncover several new mutations in Farber disease that may advance our knowledge towards establishing a genotype–phenotype correlation in this disease.

The profile of genetic alterations in four breast carcinoma cell lines, SK-BR-3, BT-474, MDA-MB361 and ZR-75-1 was examined by comparative genomic hybridization, G-band karyotyping, reverse chromosome painting and fluorescence in situ hybridization of single-copy genes. These lines are aneuploid with complex structural rearrangements and have DNA copy-number imbalances involving multiple sites that include amplification of ERBB-2 and MYC proto-oncogenes which are implicated in breast cancer pathogenesis. A novel site of high level amplification was mapped on chromosome 15. All lines were tumorigenic in nude mice, however, the latency and the incidence of tumor formation varied; SK-BR-3 and MDA-MB361 produced tumors in a shorter time and had a higher total number of genomic imbalances compared to BT-474 and ZR-75-1 cells. Tumor cell behavior in vivo was not reflected by the rate of in vitro cell proliferation. Underrepresentation on the long arm of chromosome 18 was the sole alteration that correlated with an increased tumorigenicity. Chromosome 18q is rich in tumor suppressor genes and its loss is prevalent in primary node-positive breast tumors. Cell lines with monoclonal populations preserve the genetic characteristics of the primary tumor and their use may facilitate the detection of specific alterations associated with breast cancer progression.

Cyclins are essential activators of cyclin-dependent kinases (Cdk) which, in turn, play pivotal roles in controlling transition through cell-cycle checkpoints. Cyclin G2 is a recently discovered second member of the G-type cyclins. The two members of the G-type cyclins, cyclin G1 and cyclin G2, share high structural similarity but their function remains to be defined. Here we characterize the structure of the mouse cyclin G2 gene by first cloning and sequencing the full-length mouse cyclin G2 cDNA. The cyclin G2 cDNA was used to isolate the cyclin G2 gene from a BAC library and to establish that the gene was transcribed from eight exons spanning a total of 8604bp. The cyclin G2 gene was mapped by fluorescence in situ hybridization (FISH) to mouse chromosome 5E3.3.-F1.3. This region is syntenic to a region on human chromosome 4. The expression of cyclins G1 and G2 was examined in various tissues, but no correlation between expression patterns of the two genes was observed. However, during hepatic ontogenesis the cyclin G2 expression level decreased with age, whereas cyclin G1 expression increased. Transient expression of cyclin G2-green fluorescent protein (GFP) fusion protein in NIH3T3 cells showed that cyclin G2 is essentially a cytoplasmic protein, in contrast to the largely nuclear localization of cyclin G1. Our data suggest that, despite the close structural similarity between mouse cyclins G1 and G2, these proteins most likely perform distinct functions.

Identification of specific and primary chromosomal alterations during the course of neoplastic development is an essential part of defining the genetic basis of cancer. We have developed a transgenic mouse model for liver neoplasia in which chromosomal lesions associated with both the initial stages of the neoplastic process and the acquisition of malignancy can be analyzed. Here we analyze chromosomal alterations in 11 hepatocellular carcinomas from the c-myc/TGF-α double-transgenic mice by fluorescent in situ hybridization with whole chromosome probes, single-copy genes, and 4′-6-diamidino-2-phenylindole (DAPI-) and G-banded chromosomes and report nonrandom cytogenetic alterations associated with the tumor development. All tumors were aneuploid and exhibited nonrandom structural and numerical alterations. A balanced translocation t(5:6)(G1;F2) was identified by two-color fluorescent in situ hybridization in all tumors, and, using a genomic probe, the c-myc transgene was localized near the breakpoint on derivative chromosome der 6. Partial or complete loss of chromosome 4 was observed in all tumors with nonrandom breakage in band C2. Deletions of chromosome 1 were observed in 80% of the tumors, with the most frequent deletion at the border of bands C4 and C5. An entire copy of chromosome 7 was lost in 80% of the tumors cells. Eighty-five percent of the tumor cells had lost one copy of chromosome 12, and the most common breakpoint on chromosome 12 occurred at band D3 (28%). A copy of chromosome 14 was lost in 72%, and band 14E1 was deleted in 32% of the tumor cells. The X chromosome was lost in the majority of the tumor cells. The most frequent deletion on the X chromosome involved band F1. We have previously shown that breakages of chromosomes 1, 6, 7, and 12 were observed before the appearance of morphologically distinct neoplastic liver lesions in this transgenic mouse model. Thus breakpoints on chromosome 4, 9, 14, and X appear to be later events in this model of liver neoplasia. This is the first study to demonstrate that specific sites of chromosomal breakage observed during a period of chromosomal instability in early stages of carcinogenesis are later involved in stable rearrangements in solid tumors. The identification of the 5;6 translocation in all of the tumors has a special significance, being the first balanced translocation reported in human and mouse hepatocellular carcinoma and having the breakpoint near a tumor susceptibility gene and myc transgene site of integration. Moreover, its early occurrence indicates that this is a primary and relevant alteration to the initiation of the neoplastic process. In addition, the concordance between the breakpoints observed during the early dysplastic stage of hepatocarcinogenesis and the stable deletions of chromosomes 1, 4, 6, 7, 9, and 12 in the tumors provides evidence for preferential site of genetic changes in hepatocarcinogenesis.

In this report, we have identified, sequenced, and characterized the expression pattern of a novel human gene, mammaglobin B. Mammaglobin B (MGB2) is highly homologous to mammaglobin (MGB1), a previously characterized human gene whose expression is limited to the mammary epithelium and frequently up-regulated in human breast cancer cells. Based upon amino acid sequence similarities, both mammaglobin and mammaglobin B may be considered members of a larger, mammalian multigene family that includes rabbit uteroglobin, human Clara Cell 10-kDa protein (CC10), and the multimeric rat prostatein protein. Together with the human CC10 gene, mammaglobin and mammaglobin B are closely linked on human chromosome 11q13. However, despite their primary sequence similarity and close chromosomal proximity, the expression of mammaglobin and mammaglobin B is nonconcordant in both nonmalignant and neoplastic tissue.

BID is a proapoptotic member of the BCL-2 family of cell death regulators. BID shares sequence homology with other members of the family within a single alpha-helical domain, BH3. BH3 is required for BID to interact with BCL-2 and BAX, as well as for its function as a death agonist. We have isolated and characterized mouse Bid and human BID genomic clones. The sequence for BID is encoded within five exons. We used interspecific backcross analysis to localize Bid to the distal region of mouse chromosome 6 near the Atp6e locus. Fluorescence in situ hybridization analysis localized human BID to a syntenic human region, chromosome 22q11, close to the BCR-1 gene.

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.

Dipeptidyl peptidase I (DPPI) is a lysosomal cysteine protease that catalyzes the sequential removal of dipeptides from the amino termini of various protein substrates. We have isolated a cDNA coding for murine DPPI from mouse thymus and spleen cDNA libraries. The deduced amino acid sequence codes for a protein of 462 amino acid residues; comparison of this deduced sequence with that of rat and human DPPI revealed 90.1% and 77.8% identity, respectively. Using DPPI cDNA, we obtained two BAC (Bacterial Artificial Chromosome) clones that contained the murine DPPI locus. The DPPI gene consists of seven exons and 6 introns, and spans approximately 20 kilobases. Using fluorescence in situ chromosome hybridization, we localized murine DPPI to chromosome 7D3-E1.1. We determined that DPPI protein is widely distributed in mouse tissues, although its relative abundance varies from tissue to tissue. In contrast to previous reports, we show here that DPPI mRNA and protein levels and enzymatic activity are unchanged during in vitro T cell activation, implying that this enzyme is not rate-limiting for granzyme processing.

Human uteroglobin (hUG) or Clara cell 10-kD protein (cc10 kDa) is a steroid-dependent, immunomodulatory, cytokine-like protein. It is secreted by mucosal epithelial cells of all vertebrates studied. The cDNA encoding hUG and the 5' promoter region of the gene have been characterized previously. Here, we report that the structure of the entire hUG gene is virtually identical to those of rabbit, rat, and mouse. It is localized on human chromosome 11q12.3-13.1, a region in which several important candidate disease genes have been mapped by linkage analyses. Our data indicate that candidate genes for atopic (allergic) asthma and Best's vitelliform macular dystrophy are in closest proximity to the hUG gene. To determine whether hUG gene mutation may be involved in the pathogenesis of these diseases, we studied two isolated groups of patients, each afflicted with either atopy or Best's disease, respectively. We detected a single base-pair change in the hUG gene in Best's disease patients and normal controls but no such change was detected in atopy patients. This alteration in hUG gene-sequence in Best disease family appears to be a polymorphism. Although the results of our investigation did not uncover mutations in hUG gene that could be causally related to the pathogenesis of either of these diseases, its conservation throughout vertebrate phyla implies that this gene is of physiological importance. Moreover, the close proximity of this gene to several candidate disease genes makes it an important chromosomal marker in cloning and characterization of those genes.

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 been shown to have normal diploid characteristics, are hyperplastic, and depend 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 or nonrandom and if such changes contribute to the pathogenesis of KS. Methods : We used the conventional G-banding technique and fluorescence in situ 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 exhibit 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 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 of cytogenetic markers, thus facilitating generation of specific molecular probes for detecting neoplastic cells early in the disease process.

The human histone H2A.X gene is unusual in that its transcripts are alternatively processed to yield two species, one a 0.6-kb replication-linked histone mRNA and the other a 1.6-kb polyadenylated mRNA. The H2A.X gene has been localized by fluorescence in situ hybridization to chromosome 11q23.2-q23.3, away from the known clusters of human histone genes on chromosomes 1, 6, and 12. Assignment to chromosome 11 was substantiated by analysis of human-hamster somatic cell hybrid lines. As this work was being completed, an 89-bps sequence overlap was found between the downstream regions of the H2A.X gene and the recently sequenced hydroxymethylbilane (HMB)-synthase gene. The H2A.X and HMB-synthase genes have an unusual arrangement, being transcribed towards each other with their polyadenylation sites 330 bp apart. In addition the HMB-synthase gene contains constitutive and erythroid specific promoters. K562, an erythroid cell line, was found to contain a high concentration of the 1.6-kb polyadenylated H2A.X mRNA.