INPP4B is an oncogenic regulator in human colon cancer.

Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and is a tumor suppressor in some types of cancers. However, we have found that it is frequently upregulated in human colon cancer cells. Here we show that silencing of INPP4B blocks activation of Akt and serum- and glucocorticoid-regulated kinase 3 (SGK3), inhibits colon cancer cell proliferation and retards colon cancer xenograft growth. Conversely, overexpression of INPP4B increases proliferation and triggers anchorage-independent growth of normal colon epithelial cells. Moreover, we demonstrate that the effect of INPP4B on Akt and SGK3 is associated with inactivation of phosphate and tensin homolog through its protein phosphatase activity and that the increase in INPP4B is due to Ets-1-mediated transcriptional upregulation in colon cancer cells. Collectively, these results suggest that INPP4B may function as an oncogenic driver in colon cancer, with potential implications for targeting INPP4B as a novel approach to treat this disease.

A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse.

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.

Comparison of long-term survival of cytomegalovirus promotre versus Rous Sarcoma virus promoter-driven thymidine kinase gene therapy in nude mice bearing human ovarian cancer.

The cytomegalovirus (CMV) promoter is considered one of the strongest positive regulators leading to expression of higher levels of the thymidine kinase (TK) enzyme than the Rous Sarcoma virus (RSV) promoter in vitro and in vivo. Cell killing efficacy of adenovirus-mediated CMV promoter-driven herpes simplex virus (HSV) TK gene therapy has been found to be 2 to 10 times more effective than RSV driven HSV-TK gene therapy in vitro. In this study the impact of CMV- versus RSV-driven HSV-TK gene therapy on long-term survival of nude mice bearing human ovarian cancer has been evaluated using a prospective randomized experimental design. The experiment was designed to show significance of survival differences from a 50% increase of survived days at a p-value of 0.05 with a power of 80%. All treatment groups showed an increase in median survival compared with control groups. Treatment benefit was ADV/CMV-TK vector dose dependent. At a given viral dose, no significant prolongation of survival was observed comparing CMV- and RSV-driven ADV-TK indicating that simply increasing cell killing efficacy in vitro above a minimal threshold level using a stronger promoter may not lead to prolongation of survival in the HSV-TK/GCV system.

Adenovirus mediated thymidine kinase gene therapy may enhance sensitivity of ovarian cancer cells to chemotherapeutic agents.

The current treatment concept of ovarian cancer consists of radical surgery with subsequent chemotherapy. We have shown that adenovirus (ADV) mediated thymidine kinase (TK) gene transduction of cisplatin-resistant human ovarian cancer xenotransplanted into nude mice followed by ganciclovir (GCV) administration leads to prolongation of survival or cure. In this study the interaction of ADV-TK gene therapy and selected chemotherapeutic agents commonly used for the treatment of ovarian cancer was investigated in three ovarian cancer cell lines with different growth patterns. Toxicity and cell killing efficacy of gene therapy, chemotherapy and their combinations with different concentrations and time intervals were measured by a 3-(4,5- dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) based assay. A slightly increased resistance to gene therapy was observed in cells pretreated with chemotherapy. Removal of the drugs restored the previous susceptibility of the cells to gene therapy. No antagonism was observed with gene therapy followed by chemotherapy. The concomitant application of gene therapy and chemotherapy resulted in a higher rate of cell death than the interval therapy. A dose dependent synergistic interaction was observed only for the combination of gene therapy and the topoisomerase 1 inhibitor topotecan. This synergistic effect was still seen even if the chemotherapeutic agent was added 72 hours later. Our data demonstrate that in addition to its own therapeutic efficacy, ADV-TK based gene therapy may enhance the effect of subsequent chemotherapy while up-front chemotherapy was disadvantageous.

Telomere length in human white blood cells remains constant with age and is shorter in breast cancer patients.

BACKGROUND: Telomeres, which are TTAGGG repeats at the end of the eukaryotic chromosome, are essential for complete DNA replication. Telomere length has been reported to decrease in peripheral WBC, unlike the telomerase activity found in these cells. The purpose of this study was to investigate whether telomere length in WBC is indeed age dependent and could serve as a genetic marker in breast or ovarian cancer. METHODS: Five age groups: 20-29; 30-39; 4049; 50-59 and > or = 60 years were examined. The cancer patients were 18 women with ovarian cancer and 18 women with breast cancer. Southern blot analysis of the DNA from peripheral white blood cells (WBC) was performed using 32P-labeled (TTAGGG)3 probe. Blots were scanned in a phosphoimager and analyzed by computer-assisted image analysis. RESULTS: No statistically significant correlation was observed between telomere length and age in either healthy females or cancer patients. However, significantly shorter median telomere length was found in WBC obtained from breast cancer patients as compared to healthy individuals and ovarian cancer patients. CONCLUSIONS: It is concluded that telomere length in WBC is not age dependent, but is significantly shorter in breast cancer patients.

Improvement of gene therapy for ovarian cancer by using acyclovir instead of ganciclovir in adenovirus mediated thymidine kinase gene therapy.

Adenovirus(ADV) mediated thymidine kinase(TK) gene therapy followed by ganciclovir(GCV) administration is widely used in different types of cancer. ACV shares the same mechanism of selective cell killing in ADV/TK positive cells as GCV and can be used at 4.5 times higher doses in patients without significant side effects. An increased dose of TK substrate is associated with improved bystander effect and more efficient cell killing. Toxicity and cell killing efficacy were assessed using a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide(MTT) based assay in three ovarian cancer cell lines with different proliferation patterns. At the same concentration, equal or higher cell killing efficacy and bystander effect were observed using ACV rather than GCV. 2.5 and 5 times (25 micrograms/ml and 50 micrograms/ml) higher concentrations of ACV always resulted in more effective cell killing than GCV (10 micrograms/ml, P < 0.01). Our data indicate that replacing GCV with ACV in the ADV-TK gene therapy may increase the treatment effect without increasing toxicity.

The efficacy of adenovirus-mediated gene therapy of ovarian cancer is enhanced by using the cytomegalovirus promoter.

The cytomegalovirus(CMV) promoter is considered one of the strongest positive regulators. In this study toxicity, cell killing efficacy and bystander effect of Rous Sarcoma Virus(RSV) driven herpes simplex thymidine kinase(TK) gene therapy was compared with CMV driven TK gene therapy in three ovarian cancer cell lines with different growth patterns using a 3-(4,5-dimethylthiazol)-2,5-diphenyl tetra-zolium bromide (MTT) based assay. ADV/CMV-TK was shown to be 2 to 10 times more effective in tumor cell killing than ADV/RSV-TK. The difference in cell killing efficacy between ADV/CMV-TK and ADV/RSV-TK was dependent on the individual cell line. A CMV promoter dependent eight to ten fold improvement in cell killing efficacy was observed in the relatively slow growing SKOV3 cell line which is not easily transducible, while only a 2 to 4 fold difference was observed in the easily transducible OV-CA-2774 and OV-CA-1225 cell lines. ADV/CMV-TK also showed a stronger bystander effect than ADV/RSV-TK in all three ovarian cancer cell lines. Our data demonstrated that the efficacy of adenovirus-mediated gene therapy of ovarian cancer can be enhanced by using the CMV promoter without increasing toxicity.

Cloning, expression analysis and chromosomal localization of the human nuclear receptor gene GCNF.

Germ cell nuclear factor (GCNF) is an orphan member of the nuclear receptor gene superfamily. We report the cloning of a cDNA encoding a new variant of human GCNF from human testis and its expression analysis. Southern blot analysis of the human genomic DNA indicates that the GCNF gene is not closely related to other members within the nuclear receptor superfamily. Chromosomal localization of the GCNF gene shows that the gene is located on chromosome 9 at the locus q33-34.1. In situ hybridization analysis of GCNF expression in the testis shows that human GCNF is expressed exclusively in germ cells.

Human epithelial ovarian cancer xenotransplants into nude mice can be cured by adenovirus-mediated thymidine kinase gene therapy.

BACKGROUND: Adenovirus-mediated suicide gene therapy of ovarian cancer has effective anti-tumor effects in vitro and in vivo. By transduction of ovarian adenocarcinoma with the Herpes Simplex Thymidine Kinase gene and subsequent treatment with the antiviral agent ganciclovir, prolongation of survival has been described in nude mice. So far, however, in animal models of solid tumors no cures have been reported after gene therapy. METHODS: In a prospective randomized experimental design 76 mice with xenotransplanted serous ovarian carcinoma were treated with three different doses of ADV/RSV-TK at three different time points followed by intraperitoneal ganciclovir administration. The experiment was designed to show significance of survival differences upon doubling of the number of survived days at a p-value of 0.05 with a power of 80%. The endpoint of the trial was survival. RESULTS: Treatment response was seen in all treated animals evident by significant prolongation of survival. Treatment response was dependent on the therapeutic viral dose and the tumor burden of the animal at the time of treatment. Two out of eight mice with early disease have now survived ten months without evidence of disease with untreated animals dying after nineteen days. Subcutaneous tumor development at the injection site was the reason of death in the remaining six mice of this group. CONCLUSIONS: Intraperitoneal ADV/RSV-TK suicide gene therapy of epithelial ovarian cancer in combination with ganciclovir administration can cure nude mice with early disease. This treatment modality may lend itself to incorporation into the current treatment concept of human ovarian malignancy. Clinical trials are warranted.

In vivo gene therapy of ovarian cancer by adenovirus-mediated thymidine kinase gene transduction and ganciclovir administration.

Efficacy and toxicity of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene followed by administration of ganciclovir were studied in vivo. A human epithelial ovarian cancer animal model was established in nude mice using the serous ovarian adenocarcinoma cell line Ov-ca-2774. Intraperitoneal (ip) injection of 1 x 10(8) Ov-ca-2774 cells resulted in tumor growth and formation of malignant ascites in all 15 animals. In a prospective randomized experimental design mice were treated 1, 3, or 7 days after ip injection of 1 x 10(8) cells with ip injection of 2 x 10(8), 6.7 x 10(8), or 2 x 10(9) pfu ADV.RSV-TK followed by administration of ganciclovir (10 microgram /ml, ip, bid) for 6 consecutive days. End points were survival and toxicity. Mice treated with GCV or HSV-TK alone died from 14.4 +/- 1.7 to 19.5 +/- 3.5 days after treatment as did untreated controls. No toxicity of ADV.RSV-TK was found up to 2 x 10(9) pfu (2 x 10(11) particles). The mice with the highest tumor burden treated with the lowest viral dose lived significantly longer than controls (P < 0.05). Median survival in all other groups of mice treated with ADV.RSV-TK plus GCV was even longer (P < 0.01). Treatment benefit was dependent on ADV/RSV-TK dose and tumor burden. Adenovirus-mediated thymidine kinase gene therapy is a realistic approach to ovarian cancer treatment that warrants investigation in the clinical setting.