Sensitivity Of Human Tumor Cells Research Articles

Role of culture conditions and exposure duration in determining sensitivity of human bone marrow progenitor cells to methotrexate.

The effect of drug concentration, exposure duration, and culture conditions on the cytotoxic activity of methotrexate (MTX) on normal granulocyte-macrophage colony-forming units in culture (GM-CFUC) was studied using a bilayer soft agar system with nucleoside-free medium. The degree of inhibition of colony formation depended on the type of serum supplementation. A 1 h or 2 h pulse treatment with 2 X 10(-4) M (100 micrograms/ml) MTX failed to kill GM-CFUC, when the cells were subsequently plated in a system containing 15% undialyzed fetal bovine serum (FBS). For continuous exposure the observed LD50 of MTX in the agar system was higher than 10(-4) M for 15% undialyzed FBS, 10(-5) M for 15% dialyzed FBS plus 0.25% undialyzed FBS, 10(-6) M for 15% dialyzed FBS, and 10(-8) M for 15% undialyzed horse serum. The difference for dialyzed FBS versus horse serum can be explained by differences in nucleoside concentrations. The difference for dialyzed FBS versus horse serum may be secondary to an enhancer of MTX in horse serum. For studying MTX sensitivity of human tumor cells in vitro, we suggest testing conditions that lie within the dose survival curve of GM-CFUC.

Effects of DFMO-induced polyamine depletion on human tumor cell sensitivity to antineoplastic DNA-crosslinking drugs.

We investigated the effect of pretreatment with difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, on the cytocidal responses of four human adenocarcinoma cell lines to two alkylating and crosslinking agents: chlorambucil and N,N',N"-triethylenethiophosphoramide (thiotepa). The cell lines studied included HuTu-80 (duodenum), HT-29 (colon), ME-180 (cervix), and A-427 (lung). A 48- to 72-h pretreatment with DFMO reduced intracellular putrescine and spermidine contents to less than 10% and less than 1% of control levels. This treatment also caused a 30%-70% decline in spermine content. Survival of control and DFMO-pretreated cells after treatment with chlorambucil or thiotepa was measured by a plating efficiency assay. For three of the four lines studied, the DFMO-induced partial polyamine depletion significantly protected cells from the lethal effects of chlorambucil. In ME-180 cultures alone, DFMO pretreatment did not alter the cytocidal efficacy of chlorambucil. Addition of exogenous putrescine to cultures of HuTu-80, HT-29, or A-427 24 h after DFMO addition but 24 h before treatment with chlorambucil reversed the polyamine depletion and its protective effects on chlorambucil-induced cell kill. In contrast to the above observations, DFMO and partial polyamine depletion had no effect on cell survival after thiotepa treatment for any of the cell lines investigated.

Growth of Human Renal Carcinoma in Soft Agar Colony Formation Assays Measured by Computer-Assisted Volume Analysis

Technical methods for assessing the growth and chemotherapy sensitivity of human tumor cells growing in soft-agar culture have been less than ideal. Within the past year, there have been reports of studying the extent of growth of human tumor cells in these cultures by quantitating the change in cumulative volume for the growth units observed. The present report describes the results of computer-assisted volume analysis applied to soft-agar cultures of cells from 74 primary renal cell carcinomas, 14 primary transitional cell carcinomas of the renal pelvis, and four different human renal cell carcinoma xenografts. The extent of growth in vitro observed for cells from freshly excised human renal tumors showed the expected and statistically significant relationship to tumor grade and stage. The renal cell carcinoma xenografts proliferated to a much greater extent in vitro than the cells from freshly excised human renal carcinomas. The fundamental growth limit of 109μm.3 cumulative growth unit volume per plate was confirmed by this series of experiments. Computer-assisted volume analysis appears to be a useful method to study the growth of freshly excised human renal carcinoma cells in vitro.

Effects of Lonidamine on murine and human tumor cells in vitro. A morphological and biochemical study.

Lonidamine induces in murine and human tumor cells severe morphological damage of the mitochondria and other cytoplasmic structures both 'in vitro' and 'in vivo'. Biochemical studies have demonstrated that the drug decreases oxygen consumption and lactate production. The sensitivity of human tumor cells is not related to their histotype. Lonidamine's effects on mitochondria, glycolysis, pentose phosphate pathway, and aromatase activity are discussed.

Modification of the radiation sensitivity of human tumour cells by a bis-benzimidazole derivative.

A comparison was made of the ability of either X-radiation or a DNA-specific ligand (the vital bis-benzimidazole dye; Hoechst 33342) to induce: cell killing, inhibition of de novo DNA synthesis, DNA strand breakage and the delay of cell division in human colon adenocarcinoma cells in vitro. Unlike radiation-induced cell killing, ligand-induced cytotoxicity appeared to be positively correlated with the extent of inhibition of de novo DNA synthesis--a feature consistent with the persistent binding of ligand molecules to nuclear DNA. Ligand-induced DNA strand-breaks disappeared slowly although ligand-treated cells retained apparently normal capacities to repair discrete radiogenic DNA strand-breaks. Pre-treatment of cells with Hoechst 33342 resulted in a dose-modifying enhancement of radiation resistance not associated with altered dosimetry for strand-break induction. However, radioresistance was accompanied by the protracted retention of cells in the G2 phase of the cell cycle. We suggest that the results provide direct evidence that the retention of cells in G2 phase is a sparing phenomenon and is triggered by the responses of chromatin domains to the presence of DNA damage. Our results have implications for the use of DNA-interactive agents in combined modalities for tumour therapy, and indicate a possible basis for the sparing of some tumour cells in dividing populations.

Properties of anticancer agents relevant to in vitro determinations of human tumor cell sensitivity

The physical properties of 59 anticancer agents have been examined with respect to solubility in tissue culture media, binding to ultrafiltration materials, and molecular absorbance and fluorescence behavior. Methods for dissolving these agents, which are compatible with in vitro sensitivity testing of human tumor cells to anticancer agents, are reported in this paper. The potential for anticancer agent binding to cellulose nitrate/cellulose acetate and teflon membrane ultrafilters was documented, and quantitation of these anticancer agents based upon absorbance and fluorescence spectroscopy was performed. Post-filtration quantitation of anticancer agents was found to be a reliable method for determining the actual drug concentrations available in tumor cell sensitivity testing in vitro. The properties documented herein are pharmacologically relevant parameters related to in vitro determinations of human tumor cell sensitivity to anticancer agents.

Pharmacologic requirements for obtaining sensitization of human tumor cells in vitro to combined 5-fluorouracil or ftorafur and X rays

The combined effects of X ray and 5-Fluorouracil (5-FU) in tissue culture have been studied using two human adenocarcinoma lines (HeLa and HT-29 cells). Both showed similar sensitivities to 5-FU, HeLa cells appearing somewhat more resistant to higher concentrations. Combined treatment of both cell types with 5-FU and X rays led to a time-dependent enhancement of cell killing (“radiosensitization”). Only post-radiation incubation had any effect, prior exposure to 5-FU being strictly additive. Enhanced cell killing by combined 5-FU and X rays could not be explained by either the infliction of additional acute damage in the immediate post-radiation period or an inhibitory effect of 5-FU on the repair of sub-lethal X ray injury. Rather, the enhanced cytotoxicity proved to be dependent on a damage expressed in time periods exceeding the duration of a cell doubling time in vitro. Overall, the data equally suggest that X rays may sensitize the cells to 5-FU. The enhanced cell killing is maximized if the cells are continuously exposed to 5-FU for 48 hours following the X ray exposure. These results indicate that clinical treatment regimens might be useful in evaluating 5-FU infusional scheduling in accordance with these unique requirements, which are not met by conventional bolus 5-FU and X ray therapy fractionation regimens. Ftorafur, a drug proposd to act as a slow release form of 5-FU, was found to show limited cytotoxic potential in vitro and did not significantly enhance cell killing after X ray exposure.

In vitro sensitivity of human tumor cells to spontaneous cell mediated cytotoxicity (SCMC): M. Fopp, M.E. Weber, H.J. Senn, Department of Medicine, Division of Hematology and Oncology, Kantonsspital St-Gallen, Switzerland

In vitro sensitivity of human tumor cells to spontaneous cell mediated cytotoxicity (SCMC): M. Fopp, M.E. Weber, H.J. Senn, Department of Medicine, Division of Hematology and Oncology, Kantonsspital St-Gallen, Switzerland

Clonogenic cell survival in cryopreserved human tumour cells

Cells from 3 human tumours have been grown in soft agar contained in Millipore diffusion chambers and implanted i.p. in mice. Clonal growth was obtained from fresh biopsy samples, from cryopreserved tissue, and from xenografts of the tissues in immune-suppressed mice. The radiosensitivities of a melanoma and an ovarian carcinoma were evaluated by in vitro irradiation before assay for colony formation. Xenografting did not modify the radiosensitivity of the melanoma. Cells from another tumour were exposed to Adriamycin or cyclophosphamide whilst contained within i.p. diffusion chambers; the sensitivity was similar for cryopreserved and xenografted cells. The results encourage further attempts to quantify the sensitivity of human tumour cells by these methods.

Relationship between Surviving Fractions Using the Colony Method, the LD 50 , and the Growth Delay after Irradiation of Human Melanoma Cells Grown as Multicellular Spheroids

Survival of human melanoma Na 11 cells after ..gamma.. irradiation was measured using three different endpoints: clonogenicity of single cells isolated from spheroids either before or after treatment, probability of cure by sterilization of 50% of the spheroids, and growth delay. No significant difference in radiosensitivity was found between single cells and cells from spheroids. A close relationship was found between the cell yield and the theoretical number of cells per spheroid. Good agreement was found between the LD/sub 50/ survival curve and that from monodispersed cells. When growth delay was plotted against survival a similar conclusion was reached, but with somewhat less precision, indicating that other factors must contribute to regrowth. The multicellular spheroid culture, which permits multiendpoint evaluation of survival, should provide a valid assay of human tumor cell sensitivity to radiation and chemotherapeutic treatments.

X-ray sensitivity of human tumor cells in vitro

Clonally-derived cells from ten human malignant tumors considered radiocurable (breast, neuroblastoma, medulloblastoma) or non-radiocurable (osteosarcoma, hypernephroma, glioblastoma, melanoma) were studied in cell culture and their in vitro x-ray survival curve parameters determined ( n , D o). There were no significant differences among the tumor cell lines suggesting that survival parameters in vitro do not explain differences in clinical radiocurability. Preliminary investigation with density inhibited human tumor cells indicate that such an approach may yield information regarding inherent cellular differences in radiocurability.