Hideshi Ishii

FHIT gene therapy prevents tumor development in Fhit-deficient mice

The tumor suppressor gene FHIT spans a common fragile site and is highly susceptible to environmental carcinogens. FHIT inactivation and loss of expression is found in a large fraction of premaligant and malignant lesions. In this study, we were able to inhibit tumor development by oral gene transfer, using adenoviral or adenoassociated viral vectors expressing the human FHIT gene, in heterozygous Fhit+/− knockout mice, that are prone to tumor development after carcinogen exposure. We therefore suggest that FHIT gene therapy could be a novel clinical approach not only in treatment of early stages of cancer, but also in prevention of human cancer.

Interactions in the Error-prone Postreplication Repair Proteins hREV1, hREV3, and hREV7

Most mutations after DNA damage in yeastSaccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase ζ that consists of scRev3 and scRev7 proteins. Recently, the humanREV1 (hREV1) and REV3(hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts. Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown. We report here the analyses of precise interactions in the human REV proteins. The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays. The homodimerization of hREV7 was also detected in the two-hybrid analysis. In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro. These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage.

Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppresses tumorigenicity in lung and cervical cancer cell lines

Loss of expression of the Fhit protein is often associated with the development of many human epithelial cancers, including lung and cervical carcinomas. Restoration of Fhit expression in cell lines derived from these tumors has however yielded conflicting results, prompting the need for careful evaluation of the oncosuppressive potential of FHIT. In the present study, we have investigated the effect of Fhit reintroduction in seven lung cancer and three cervical cancer cell lines. To achieve efficient gene transfer and high levels of transgene expression, we have used an adenoviral vector to transduce the FHIT gene. The induction of apoptosis was evaluated by using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and propidium iodide staining. Activation of caspases was detected by using Western blot analysis, and tumorigenic potential of transduced cells in the nude mouse was also assessed. Restoration of Fhit expression induced apoptosis in all Fhit-negative cell lines, with Calu-1, H460, and A549 being the most susceptible among the lung cancer cell lines and SiHa cells among cervical carcinomas. Activation of caspase-8 was always associated with Fhit-mediated apoptosis, and in vivo tumorigenicity was either abolished by FHIT gene transfer (in H460 and SK-Mes cells) or strongly suppressed (in A549 and SiHa cells). Our data demonstrate oncosuppressive properties and strong proapoptotic activity of the Fhit protein in lung and cervical cancer cell lines and strengthens the hypothesis of its possible use as a therapeutic tool.

A human REV7 homolog that interacts with the polymerase zeta catalytic subunit hREV3 and the spindle assembly checkpoint protein hMAD2.

Widespread alteration of the genomic DNA is a hallmark of tumors, and alteration of genes involved in DNA maintenance have been shown to contribute to the tumorigenic process. The DNA polymerase ζ of Saccharomyces cerevisiae is required for error-prone repair following DNA damage and consists of a complex between three proteins, scRev1, scRev3, and scRev7. Here we describe a candidate human homolog of S. cerevisiae Rev7 (hREV7), which was identified in a yeast two-hybrid screen using the human homolog of S. cerevisiae Rev3 (hREV3). The hREV7 gene product displays 23% identity and 53% similarity with scREV7, as well as 23% identity and 54% similarity with the human mitotic checkpoint protein hMAD2. hREV7 is located on human chromosome 1p36 in a region of high loss of heterozygosity in human tumors, although no alterations of hREV3 or hREV7 were found in primary human tumors or human tumor cell lines. The interaction domain between hREV3 and hREV7 was determined and suggests that hREV7 probably functions with hREV3 in the human DNA polymerase ζ complex. In addition, we have identified an interaction between hREV7 and hMAD2 but not hMAD1. While overexpression of hREV7 does not lead to cell cycle arrest, we entertain the possibility that it may act as an adapter between DNA repair and the spindle assembly checkpoint.

Regression of upper gastric cancer in mice by FHIT gene delivery

Fhit expression is reduced in most cancers, and Fhit replacement by FHIT expression viruses in lung, esophageal, pancreatic, and cervical cancers induces apoptosis in the cancer cells. Mice carrying one or two inactivated Fhit alleles are hypersensitive to development of N‐nitrosomethylbenzylamine (NMBA)‐induced forestomach tumors. In the present study, we investigated the kinetics and mechanism of tumor reversal and intervention by oral delivery of FHIT expression viruses. Tumor analysis showed that: a) by 37 days post‐NMBA, control mice showed ∼7 tumors and by 84 days ∼10 tumors/forestomach; b) mice receiving FHIT virus at 2 or 42 days post‐NMBA showed significantly reduced tumor burdens; c) Fhit was still expressed at 82 days postinfection; d) control viral infection had no effect on tumor development; and e) reduced Bcl2, increased Bax expression, and increased TUNEL‐positive apoptotic nuclei characterized the restored epithelia of FHIT transduced forestomachs. Thus, FHIT viral gene delivery prevents or retards development of carcinogen‐induced forestomach tumors and reverses development of established tumors by 60–70% through an apoptotic pathway. This dramatic reduction in tumor burden emphasizes the efficacy of targeting the FHIT apoptotic pathway for tumor eradication.

FEZ1/LZTS1 Is Down-Regulated in High-Grade Bladder Cancer, and Its Restoration Suppresses Tumorigenicity in Transitional Cell Carcinoma Cells

FEZ1/LZTS1 is a tumor suppressor gene that maps to chromosome 8p22, a chromosomal region frequently deleted in many human malignancies, including transitional cell carcinoma (TCC) of the urinary bladder. FEZ1/LZTS1 alterations have been reported in esophageal, breast, prostate, and gastric carcinomas. Fez1 expression was studied in five TCC-derived cancer cell lines by Western blot analysis and in 60 primary TCCs of the urinary bladder by immunohistochemistry. Fez1 protein was absent or reduced in four of five cell lines and in 37 of 60 primary TCC examined. We also restored Fez1 protein expression in human SW780 TCC-derived cells lacking endogenous Fez1 protein to study the effects of Fez1 expression on cell proliferation, cell kinetics, and tumorigenicity in BALB/c nude mice. In vitro transduction of SW780 Fez1-negative cell, with Ad-FEZ1, inhibited cell growth, altered cell cycle progression, and suppressed subcutaneous tumor growth in nude mice. These results suggest that FEZ1/LZTS1 gene plays a role in the development of TCC of the urinary bladder by acting as a bona fide tumor suppressor gene both in vitro and in vivo.

Collecting duct carcinoma of the kidney: an immunohistochemical study of 11 cases

BackgroundCollecting duct carcinoma (CDC) is a rare but very aggressive variant of kidney carcinoma that arises from the epithelium of Bellinis ducts, in the distal portion of the nephron. In order to gain an insight into the biology of this tumor we evaluated the expression of five genes involved in the development of renal cancer (FEZ1/LZTS1, FHIT, TP53, P27kip 1, and BCL2).MethodsWe studied eleven patients who underwent radical nephrectomy for primary CDC. All patients had an adequate clinical follow-up and none of them received any systemic therapy before surgery. The expression of the five markers for tumor initiation and/or progression were assessed by immunohistochemistry and correlated to the clinicopathological parameters, and survival by univariate analysis.ResultsResults showed that Fez1 protein expression was undetectable or substantially reduced in 7 of the 11 (64%) cases. Fhit protein was absent in three cases (27%). The overexpression of p53 protein was predominantly nuclear and detected in 4 of 11 cases (36%). Immunostaining for p27 was absent in 5 of 11 cases (45.5%). Five of the six remaining cases (90%) showed exclusively cytoplasmic protein expression, where, in the last case, p27 protein was detected in both nucleus and cytoplasm. Bcl2 expression with 100% of the tumor cells positive was observed in 4 of 11 (36%) cases. Statistical analysis showed a statistical trend (P = 0.06) between loss and reduction of Fez1 and presence of lymph node metastases.ConclusionsThese findings suggest that Fez1 may represent not only a molecular diagnostic marker but also a prognostic marker in CDC.