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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.

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 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.

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.

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.

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.

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.

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 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 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.

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.

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.

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.

In the search for specific chromosomal alterations in human hepatocellular carcinomas (HCC), we analyzed two new HCC cell lines and identified nonrandom changes by combined G-banding and fluorescence in situ hybridization (FISH). Cell line 7703 was established from an HCC deriving from a patient in the Qidong region of China, where the incidence of HCC is very high and is associated with hepatitis-B virus infection and exposure to aflatoxin. This line has a highly rearranged karyotype eliciting complex rearrangements involving the majority of chromosomes. The second line, SK-Hep-1, derived from a liver adenocarcinoma, is less heterogeneous, having few altered chromosomes. We have characterized the majority of structural and numerical alterations and identified in both lines unbalanced translocations with the breakpoints nonrandomly involving regions 1p36 and 3p14 and gain of chromosome 6p and 8q. While gain of 6p and 8q are recurrent in HCC, translocations of 1p and 3p are described for the first time. Damage and recombination at the breakpoint sites on chromosomes 1 and 3 might have resulted in activation of proto-oncogene, formation of new oncogenic chimeric genes, or loss of tumor suppressor genes. Published by Elsevier Science Inc.

Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.

Chromosomal abnormalities are the hallmark of cancer cells. Recurring and highly consistent structural and numerical alterations have been identified in a large number of leukemias, lymphomas, and solid tumors. The identification of recurrent genetic alterations and the isolation of molecular markers have clinical applications in the diagnosis and prognosis of neoplasia and in the detection of minimal residual disease that are essential for designing the most effective therapeutic approach. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) are powerful techniques for detection of genomic alterations. The battery of FISH methods and DNA probes that are available can resolve virtually any chromosomal alterations regardless of their complexity. Combined chromosome banding, multifluor or spectral karyotype, and comparative genomic hybridization (CGH) allow identification of structural and numerical alterations on a global basis, mapping of the DNA copy number on the entire tumor genome, complete derivation of complex rearrangements, and localization of the breakpoints of translocations and deletions. Regions of recurrent alterations can be microdissected, amplified, microclone libraries constructed and probes localized on extended chromosomes or chromatin fibers for construction of high resolution physical maps that are critical for positional cloning and gene identification. In this review we attempted to cover the current trends in cancer molecular cytogenetics, and to outline the importance of molecular chromosome analysis in the understanding of oncogenesis and its clinical applications.

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 target specificity of viral integration is essential to determining the biologic significance of this integration to various pathologic conditions, including cancer. In this study the chromosomal features of several human papillomavirus (HPV)-16 integration sites mapped by in situ hybridization in human keratinocyte lines were visualized directly by G-banding and differential labeling with bromodeoxyuridine of later replicating domains. G-negative chromosomal bands exhibiting late replication were selectively targeted by HPV-16, suggesting that the structural and functional relationship of the state of chromatin condensation and replication is critical in accessibility to virus integration.

Deleted in liver cancer 1 (DLC1) is a multidomain Rho-GTPase-activating protein (RhoGAP) and a tumor suppressor in several human common cancers. DLC1's structure predicts its interaction with a number of other proteins with various cellular functions. Previously we have shown that S100A10 (also known as p11), a key plasminogen receptor on the surface of cell membrane, colocalizes and binds with DLC1 in cell cytoplasm. The binding sites were mapped to the central domain of the DLC1 and at the end of C-terminus of S100A10. The same terminus is also a binding site for Annexin A2, which is believed to regulate availability of extracellular S100A10 and, therefore, dynamics of plasmin formation. Now we demonstrate that DLC1 competes with Annexin A2 for interaction with S100A10. DLC1 binding to S100A10 did not affect DLC1's RhoGAP activity but did reduce availability of S100A10 for binding by Annexin A2. Furthermore, the expression of DLC1 also affected S100A10 expression in a dose dependent manner, and increased S100A10 ubiquitin-dependent degradation. All of the above interactions attenuated plasminogen activation and resulted in inhibition of in vitro cell migration, invasion, colony formation and anchorage independent growth of aggressive and tumorigenic lung tumor cells. Transduction of DLC1-negative breast tumor cells significantly reduced the ability of tumor cells to form pulmonary metastases as well as inhibited distant dissemination of liver tumor cells in athymic mice. Current observations provide a plausible mechanism for DLC1 antimetastatic function by negatively affecting plasminogen activation, which in turn lead to a significant reduction in invasiveness of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2349.