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.
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.
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.
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.
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.
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-2alpha, POTE-2beta, POTE-2gamma, 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.
Human Mitochondrial Topoisomerase I Zhang, Hongliang; Barceló, Juana M.; Lee, Benson ...
Proceedings of the National Academy of Sciences - PNAS,
09/2001, Letnik:
98, Številka:
19
Journal Article
Recenzirano
Odprti dostop
Tension generated in the circular mitochondrial genome during replication and transcription points to the need for mtDNA topoisomerase activity. Here we report a 601-aa polypeptide highly homologous ...to nuclear topoisomerase I. The N-terminal domain of this novel topoisomerase contains a mitochondrial localization sequence and lacks a nuclear localization signal. Therefore, we refer to this polypeptide as top1mt. The pattern of top1mt expression matches the requirement for high mitochondrial activity in specific tissues. top1mt is a type IB topoisomerase that requires divalent metal (Ca2+or Mg2+) and alkaline pH for optimum activity. The TOP1mt gene is highly homologous to the nuclear TOP1 gene and consists of 14 exons. It is localized on human chromosome 8q24.3.
The isolation of genes involved in cancer development is critical for uncovering the molecular basis of cancer. We report here the isolation of the full-length cDNA and chromosomal localization of a ...new gene frequently deleted in liver cancer (DLC-1) that was identified by representational difference analysis. Loss of heterozygosity was detected for DLC-1 in 7 of 16 primary hepatocellular carcinomas (HCCs) and in 10 of 11 HCC cell lines. Although mRNA for DLC-1 was expressed in all normal human tissues, it was not expressed in 4 of 14 HCC cell lines. Full-length cDNA for DLC-1 of 3800 bp encodes a protein of 1091 amino acids, has 86% homology with rat p122 RhoGAP gene, and was localized by fluorescence in situ hybridization on chromosome 8 at bands p21.3-22. Deletions on the short arm of chromosome 8 are recurrent in liver, breast, lung, and prostate cancers, suggesting the presence of tumor suppressor genes. DLC-1 may be a tumor suppressor gene in liver cancer as well as in other cancers.
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 approximately 5-fold and Top2alpha compensates for Top1 deficiency. A prominent feature of the siTop1 cells is genomic instability, with chromosomal aberrations and histone gamma-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.
DLC1 (Deleted in Liver Cancer 1) gene encodes a RhoGTPase-activating protein (RhoGAP), which exerts most of its tumor suppressor functions through suppression of small Rho GTPases proteins RhoA, ...RhoB, RhoC and to some degree Cdc42, but not Rac. RhoGTPases are implicated in NF-κB activation in highly invasive prostate carcinoma (PCA), with consequences on cell proliferation, survival and metastatic capacity. Here we demonstrate that DLC1 transduction in two androgen-independent (AI) and highly metastatic PCA cell lines negatively regulates NF-κB activity in a GAP- and α-catenin-dependent manner. Expressed DLC1 protein suppresses the phosphorylation of NF-κB inhibitor, IκBα, causes its relocation from membrane ruffles into cytoplasm and attenuates its ubiquitination and subsequent degradation. DLC1-mediated NF-kB suppression and its effects are comparable to NF-κB inhibition using either shRNA knockdown or peptide inhibitor. Expression of transduced DLC1 suppressed the expression of NF-κB mediated genes. Such effects were found to be reliant on presence of calcium, indicating that the observed modifications are dependent on, and enabled by DLC-mediated stabilization of adherens junctions. These results expand the multitude of DLC1 interactions with other genes that modulate its oncosuppressive function, and may have potential therapeutic implications.
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.
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