Biomedical subjects

Thomas C Hamilton

Publications and source records attributed to Thomas C Hamilton.

21 records · Page 2Linked to original sources

PubMed2002

Activation of cancer-specific gene expression by the survivin promoter.

BACKGROUND: Survivin, a member of the IAP (inhibitor of apoptosis) gene family, appears to be overexpressed in common cancers but not in corresponding normal adult tissues. To investigate whether the survivin promoter controls cancer cell-specific gene expression, we determined whether the survivin gene promoter could regulate reporter gene expression in cancer cell lines and xenografts. METHODS: Survivin protein levels were determined in human and murine cancer cell lines and in normal tissues of adult C57BL/6 mice by Western blot analysis. A reporter construct in which a portion of the survivin gene promoter was used to drive transcription of a human secreted alkaline phosphatase (SEAP) gene was transiently transfected into cancer cells, and promoter activity was extrapolated from SEAP activity. A2780 human ovarian cancer cells were transfected with this construct, and stable transfectants were injected into the intrabursal ovarian space of immunodeficient mice. Tumor growth was monitored, and plasma SEAP levels were used as a measure of survivin promoter activity in vivo. RESULTS: Survivin protein was detected in all cancer cell lines examined but not in most normal adult mouse tissues. After transfection, the survivin promoter was more active in all cancer cell lines than in normal ovarian surface epithelial cells or mouse 3T3 cells. After 0.8 x 10(6) stable transfectant cells were injected into the intrabursal cavity of mouse ovaries, plasma SEAP activity was detected within 24 hours, and the activity increased with time and tumor growth. CONCLUSION: Transfection experiments indicate that survivin protein expression in cancer tissue appears to be regulated, at least in part, transcriptionally. Thus, the survivin promoter may be useful in controlling gene expression in cancer cells.

Alkaline Phosphatase↗

PubMed2002

Targeted gene therapy of ovarian cancer using an ovarian-specific promoter.

OBJECTIVES: The "suicide" gene therapy of cancer using promoters such as cytomegalovirus could cause severe toxicity to normal tissues due to a lack of specificity of prodrug activation. Therefore, we investigated gene therapy of ovarian cancer using ovarian-specific promoter (OSP1) to limit the synthesis of the prodrug activating enzyme HSVtk to ovarian cancer cells. METHODS: The HSVtk expressing plasmid pOSP1-HSVtk was created and transfected into an ovarian cancer cell line OVCAR3. The ganciclovir (GCV) sensitivity of the stable transfectants was evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Tissue specificity of this promoter was evaluated by comparing the sensitivity to GCV between ovarian and nonovarian cancer cell lines after they were transfected with pOSP1-HSVtk. One transfectant sensitive to GCV was implanted intraperitoneally to immunocompromised mice which were treated subsequently with GCV. Furthermore, this ovarian cancer survival model was used to evaluate the in vivo efficacy of cationic lipid mediated pOSP1-HSVtk gene delivery followed by GCV treatment. RESULTS: Stable transfectants of OVCAR3 cells bearing OSP1-HSVtk became more sensitive to GCV treatment compared to the parental cell line and vector transfected OVCAR3 cell line. OSP1-HSVtk could specifically sensitize the OVCAR3 ovarian cancer cell line to GCV. SCID mice transplanted with the OVCAR3 transfectant and treated with GCV survived longer than the mice without GCV treatment (P = 0.032). In vivo gene delivery mediated by a cationic lipid (GL67) followed by GCV treatment yielded a longer survival in the OVCAR3 survival model (P = 0.016). CONCLUSIONS: The OSP1 promoter can selectively direct suicide gene therapy of ovarian cancer and the in vivo efficacy is improved by using a cationic lipid GL67 as delivery vehicle as opposed to the direct injection of plasmid.

Female↗

PubMed2002

Decreased expression of retinol-binding proteins is associated with malignant transformation of the ovarian surface epithelium.

We have developed a modified form of suppression subtractive hybridization (SSH) that allows multiple specimens of distinct phenotypic groups to be compared for consistent differences in gene expression. We applied this system to identify genes that were expressed in normal rat ovarian surface epithelial (ROSE) cells but whose expression was lost/downregulated in four independently transformed rat ovarian cancer cell lines. Northern blot analysis using 14 of 28 nonredundant cDNA fragments from this difference library showed that the mRNA transcripts were present in normal ROSE cells but lost or markedly downregulated in four related transformed cell lines. Of particular interest, cellular retinol-binding protein 1 (CRBP1) and retinol-binding protein (RBP), two genes whose products are involved in retinol transport and metabolism, were found to be downregulated in this ovarian cancer model system. To determine if this change had relevance to human ovarian cancer, we evaluated a series of human ovarian cancer cell lines and a limited number of frozen human ovarian tumors and found lost or decreased expression of CRBP1 and RBP relative to expression in human ovarian surface epithelial (HOSE) cells. We hypothesize that the loss of CRBP1 and RBP expression disrupts retinol metabolism and retinoic acid production, which may facilitate the occurrence of genetic damage leading to the malignant transformation of the ovarian surface epithelium, the cells from which ovarian cancer arises.

Blotting, Northern↗