The objectives of this study were to quantity and compare the activities of a minimal heat shock (HS) promoter and other promoters used in gene therapy applications, and to identify strategies to amplify the heat inducibility of therapeutic genes. Human tumour cells were transiently or stably transfected with the HS promoter driving expression of reporter genes. HS promoter activity was induced transiently, with maximum activity 16-24 h after HS, and was dependent on temperature. The activity of the minimal HS promoter was similar, after 42 degrees C HS for 1 h, to that of the cytomegalovirus (CMV) promoter. To determine if the HS promoter could be used to activate a second conditional promoter, cells were transiently transfected with vectors containing both the HS and human immunodeficiency virus type 1 (HIV1) promoters. When the IL-2 gene was placed downstream of the HIV1 promoter. IL-2 production was temperature-independent. The addition of the HIV tat gene downstream of the HS promoter caused IL-2 to be induced more than 3 fold after a single 42 degrees C HS. These data indicate that the minimal HS promoter, following activation by clinically attainable temperatures (< or = 42 degrees C), can drive expression of therapeutic genes at levels comparable to the CMV promoter and be used in conjunction with a second conditional promoter to drive temperature-dependent, gene expression.

Genes, tat↗

PubMedDate not supplied

Cellular eukaryotic initiation factor 5A content as a mediator of polyamine effects on growth and apoptosis.

The polyamines are essential for eukaryotic cell growth. One of the most critical effects of polyamines on cell growth is the availability of spermidine for the post-translational modification of eIF-5A. Because hypusine-containing eIF-5A is necessary for cell proliferation, depletion of cellular polyamines suppresses growth by depleting cellular modified eIF-5A content. Excess putrescine accumulations in DH23A/b cells induces apoptosis and suppresses the formation of hypusine-containing eIF-5A. Treatment of DH23A/b cells with diaminoheptane also suppresses modified eIF-5A formation and induces apoptosis. These data suggest that suppression of modified eIF-5A formation may play a role in putrescine-induced apoptosis as well.

Animals↗

PubMedDate not supplied

Thermal dose and time-temperature factors for biological responses to heat shock.

The application of hyperthermia in human cancer therapy, especially by radiotherapists who are accustomed to prescribing ionizing radiation treatments in physical dose units, has stimulated workers in this area to consider the possibility and utility of defining a unit of 'thermal dose'. Previous thermal dose definitions have, primarily, been based on biological isoeffect response relationships, which attempt to relate exposure times that elicit a given biological response at one temperature to exposure times at another temperature that elicit the same biological response. This 'equivalent time' method is shown to have certain limitations. For both 42.4 and 45 degrees C hyperthermia, these relationships accurately describe cell survival responses only when the heating rate is rapid (greater than 0.5 degrees C min-1 from ambient to hyperthermic temperature). Further, the form of these isoeffect relationships appears to be temperature range and cell/tissue-type dependent, and it is suggested that these relationships be referred to as a 'time-temperature factor' (TTF) to help distinguish them from possible physical thermal dose definitions. Two physical dose definitions are discussed, one being simply exposure time at some temperature and the other being a more fundamental definition, the free energy change which is a temperature-dependent driving force for chemical reactions.

Animals↗

PubMedDate not supplied

Chemoprevention by difluoromethylornithine: correlation of an in vitro human cell assay with human clinical data for biomarker modulation.

The efficacy of difluoromethylornithine (DFMO) as a chemopreventive agent has been tested in vitro using a human epidermal cell (HEC) assay with growth inhibition and involucrin induction as endpoints. Suppression of polyamine content is currently being utilized as a biomarker in clinical trials for the chemopreventive efficacy of DFMO against colon cancer formation. We have now examined the effects of DFMO on suppression of polyamine content in the HEC assay. The findings indicate 1) the % change in spermidine to spermine ratio and the depletion of putrescine show excellent correlation with chemopreventive efficacy in vitro; 2) the effective concentrations in vitro overlap the plasma concentrations in the clinical trial. These observations serve as further validation of the usefulness of the HEC assay as a screen for chemopreventive efficacy.

Antineoplastic Agents↗

PubMedDate not supplied

Polyamine contents in rectal and buccal mucosae in humans treated with oral difluoromethylornithine.

Difluoromethylornithine (DFMO) is an investigational chemopreventive agent that inhibits ornithine decarboxylase (ODC) activity, lowers cellular polyamine concentrations, and decreases cell proliferation in vivo and in vitro. In five subjects we have compared the polyamine concentrations in rectal mucosal biopsies and in exfoliated buccal mucosal cells (EBM) before and after DFMO treatment to assess the suitability of EBM as an easily accessible marker tissue for DFMO suppression of polyamine synthesis in the rectal mucosa. One month of 3 g/m2/day of DFMO treatment caused a statistically significant decrease in putrescine and spermidine concentrations in rectal mucosa biopsy specimens but not in EBM samples. ODC activity in EBM was high (approximately 1 mumol/min/mg protein), resistant to DFMO inhibition (Ki = 4200 microM), dependent on GTP concentration (maximal at 0.1 mM), and was reduced concomitantly with bacterial concentration by antiseptic mouthwashing. Bacteria adherent to EBM were visible by electron microscopy. Forty bacterial colonies/ng protein were culturable from washed EBM samples. Oral bacteria preclude the use of EBM samples as a marker tissue of DFMO effect in the rectal mucosa, but oral DFMO therapy is effective in depleting polyamines in rectal mucosa.

Administration, Oral↗

PubMedDate not supplied

Ornithine decarboxylase and polyamines in colorectal neoplasia and mucosa.

Ornithine decarboxylase (ODC) and polyamines are intimately involved in normal cellular proliferation and are likely to play a role in carcinogenesis. ODC activity and polyamine content were measured in tissue samples obtained during colonoscopy from 48 benign neoplastic polyps (20 tubular adenomas; 28 villous adenomas), 18 cancers (including 5 malignant polyps), and adjacent mucosa. ODC activity in polyp and cancer tissue specimens was higher than in adjacent mucosa in 75 and 83% of pairs, respectively. Similarly, putrescine, spermidine, and spermine contents were higher in the majority of polyps and cancers compared to adjacent mucosa. ODC activity and polyamine content in colonic mucosa from 10 patients without a history of colorectal neoplasia were not different from adjacent mucosal values in the patients with neoplasia. In conclusion, ODC and polyamines are elevated in the majority of colorectal neoplasms, but amounts in normal mucosa do not differentiate between patients with cancer, benign neoplastic polyps, and normal subjects.

Adenoma↗