Karimullah A. Zirvi

Continuous growth of human tumor cell lines in serum-free media

SummaryFive human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10−10M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones influence cell growth.

α-Difluoromethylornithine inhibits liver metastasis produced by intrasplenic injection of human tumor cells into nude mice

The purpose of these studies was to examine metastatic potentials of a human colon tumor xenograft (T6) and three different human tumor cell lines (LS174T, HT29 and A549) using the intrasplenic-nude mouse model system (ISMS model system). A further objective was to study the activity ofα-difluoromethylornithine (DFMO) against primary and metastatic growth of the xenograft and the three cell lines. DFMO is an irreversible inhibitor of ornithine decarboxylase, a rate-limiting step in polyamine biosynthesis. Tumor burdens in the liver of nude mice were observed 6 weeks after the intrasplenic injection with LS174T and 12–14 weeks after intrasplenic injections with T6, HT29 and A549. Most of the mice developed primary tumor growth in the spleens. DFMO showed significant activity against liver metastases but had little or no activity against primary tumor growth in the spleens of the ISMS model and against s.c. growth of the xenograft. The studies demonstrated that the ISMS model system is an excellent system for studying metastatic behavior of human tumors and for studying the antimetastatic activity of experimental drugs.

Development of serum-free media for the growth of human gastrointestinal adenocarcinoma xenografts as primary tissue cultures

SummaryThe growth-promoting effect of several hormones and growth factors on eight human colon tumor cell lines (SW 48, SW 403, SW 480, SW 620, SW 948, HT29, LS174T and Caco-2) was studied using seven different chemically defined serum-free media [GF3: Chees essential medium plus insulin, transferrin and selenium; GF3F: GF3 plus fetuin; GF4: GF3 plus linoleic acid/ bovine-serum albumin (BSA); GF5: GF4 plus fetuin, GF5E, GF5 plus EGF; GF5T: GF5 plus triiodothyronine; GF7: GF3 plus EGF, transferrin, insulin, linoleic acid/ BSA, oleic acid/BSA and fetuin]. GF5 appears to be the best serum-free medium as it supported continuous growth of all of the colon tumor cell lines. GF5 also supported growth of five of the seven human colon and stomach tumor xenografts as primary tissue cultures. However, the stomach xenograft cells had a very slow growth rate as compared to the colon xenograft cells in the medium. Cells grown in GF5 retained their tumorigenicity in athymic (nude) mice and characteristic cellular morphology. GF7 was the poorest of all of the serum-free media studied as none of the cell lines or xenografts grew in this medium.

pH-related effects of sodium cyanate on macromolecular synthesis and tumor cell division

In past work, the selective effects of sodium cyanate on macromolecular synthesis in tumors have not been seen with cells in culture. We have explored the possibility that differences in the response of tumor cells to cyanate in vivo and in vitro may be related to the pH in the environment to which cells are exposed. When rat hepatoma (HTC) cells were incubated with sodium cyanate (0.25 mg/ml), there was a greater inhibition of precursor incorporation into RNA and DNA with a decrease in pH from 7.4 to 6.6. At pH 7.4 there was no significant effect of sodium cyanate on the incorporation of [3H]leucine into protein of rat hepatocytes and HTC cells, but at pH 6.6 there were decreases of 50% or greater. The time of response and the reversibility of the inhibitory effects of sodium cyanate were not those anticipated from carbamoylation of amino groups but were compatible with modification of sulfhydryl groups. The uptake of [14C]sodium cyanate in HTC cells and human colon cancer (HT29) cells was greater at pH 6.6 than at 7.4. Over a period of 4 days there was a slower rate of cell division by HTC and HT29 at pH 6.6 than at pH 7.4. The addition of sodium cyanate caused a further reduction in the rate of proliferation, and at a concentration of 0.25 mg sodium cyanate/ml there were decreases in cell numbers. The data suggested that a lower interstitial pH in tumors than normal tissues would result in greater sensitivity to inhibitory effects of sodium cyanate on macromolecular synthesis.

Activity of cancer chemotherapeutic agents against human colorectal carcinomas grown as primary tissue culture

SummaryImproved procedures are described for the seeding of primary cultures from human colon adenocarcinoma and for the use of these cultures in the evaluation of drug effects. Two of the specimens studied were xenografts maintained in athymic (nude) mice, while the other six were biopsies obtained directly from patients. Tumor cells obtained directly from the patients proliferated in defined hormone-supplemented medium to the exclusion of other cells.In drug-response studies with cultures from a colon tumor biopsy all four drugs studied (4′-deoxydoxorubicin, 4′-O-methyldoxorubicin, 5-fluorouracil, and 1,3-bis-[chloroethyl]-1-nitrosourea) inhibited growth of the cells within 3–6 days after drug treatment. On an equitoxic dose basis (LD10 in mice), 4′-deoxydoxorubicin appeared to be the most active drug. This drug also showed dose-dependent activity against one of the xenografted tumors in vitro. In dose-response studies with cultures from another patients colon tumor, doxorubicin and 5-fluorouracil showed significant activity against the tumor 10 days after the drug treatment with concentrations at 1X and 10X average peak plasma levels.

Influence of pH on the modification of thiols by carbamoylating agents and effects on glutathione levels in normal and neoplastic cells

SummaryIn previous studies, we have suggested that the selective inhibitory effect of sodium cyanate (NaOCN) on hepatoma metabolism may be due to the lower pH observed in tumors relative to normal tissues. Lower pH might enhance the action of NaOCN by increasing the formation of isocyanic acid and carbamoylation of sulfhydryl groups. In the present work, studies were conducted on the effect of pH on the carbamoylation of sulfhydryl groups. The data indicated that carbamoylation of the sulfhydryl group of glutathione by NaOCN was enhanced by decreasing the pH from 7.4 to 6.6. A less pH-dependent response was observed with organic isocyanates. However, all reactions were reversible after the pH was increased by the addition of base. Kinetic studies showed that the rate of the reaction is very rapid, a maximal effect occurring within the first 10 min. Dose-dependent modifications of cellular glutathione by NaOCN and organic isocyanates were observed in human HT29 colon tumor cells, rat HTC hepatoma cells, and rat hepatocytes. The rate of carbamoylation of the glutathione sulfhydryl group in cells was similar to that of pure glutathione (GSH). The effect of buthionine sulfoximine on GSH levels in cells was at least as great as that of sodium cyanate, but only the latter showed inhibitory effects on macromolecular synthesis; these were very rapid, pH-dependent, and reversible in tumor cells. Our results suggest that cellular sulfhydryl group(s) other than that of GSH might be involved in the effect of NaOCN on macromolecular synthesis.

Combined effect of pH and sodium cyanate on the inhibition of tumor cell proliferation and metabolism by BCNU and hyperthermia

SummaryIn previous studies, we have found that combined treatment with BCNU and sodium cyanate could have a greater effect on the survival of mice bearing B16 melanoma than treatment with either agent alone. With rat hepatoma and human colon cancer cells in culture, we have obtained evidence that the inhibition of cell proliferation by sodium cyanate is greater at pH 6.6 than at pH 7.4. In the present work, the effects of combination treatments on the proliferation of cancer cells were studied with cyanate, pH, BCNU, and hyperthermia. With HT29 human colon cancer cells, the inhibitory effect of BCNU (50–100 µg/ml) was greater when the cells were treated at pH 6.6 than at pH 7.4. The influence of pH appeared to be absent or minimal at lower or higher concentrations of BCNU. We confirmed our previous observation that the inhibition of proliferation of LS174T human colon cancer cells is greater at pH 6.6 than at pH 7.4, and we observed an inhibitory effect of BCNU (50 or 200 µg/ml). However, no more than additive effects were seen with combination treatment. An inhibitory effect of hyperthermia was seen for the incorporation of [3H]-leucine into protein of rat hepatoma cells (HTC) and for that of [3H]-thymidine into DNA of human colon cancer (HT29) cells. In neither case was the effect of hyperthermia significantly enhanced by treatment with sodium cyanate beyond that seen with one of the treatments alone. The data confirmed that the inhibitory effect of sodium cyanate on cell proliferation can be enhanced by a low pH but did not provide evidence for synergistic effects in combination with BCNU or hyperthermia.

Tumorigenicity of EJ-ras oncogene transformed NIH 3T3 cells and expression of plasminogen activators

A number of clonal cell lines have been isolated from NIH 3T3 cells transfected with the plasmid, pSV2 gpt-EJ-ras. The plasmid expresses Val12 instead of Gly12 in p21 ras protein and can be selected for the expression of E. coli XGPRT gene in mammalian cells. Southern analyses of the Eco R1 and Bam H1 digests of chromosomal DNA shows that multiple copies of the plasmid are integrated in a tandem sequence in the clones used in this study. The transfectants showed refractile appearance and criss-crossed pattern of growth, exhibited elevated expression of ras mRNA and formed tumors in nude mice commensurate with the copy number of the integrated EJ-ras gene. The increased propensity to form tumors did not correlate with the expression of urinary or tissue plasminogen activators (u-PA or t-PA). The cellular and secreted activity of u-PA in fact decreased as the ras gene expression increased. These data show that the enhanced tumorigenicity of transformed murine cells is related to the tandem integration and expression of human EJ-ras. The overexpression of ras has very little effect on t-PA but appears to suppress u-PA activity.

Comparative Analysis of Cellular and Extracellular Proteins Secreted by Two Human Tumor Cell Lines

Two human tumor cell lines, melanoma (TWI) and colon carcinoma (HT-29), replicate continuously in serum-free C-ITS medium [Chees essential medium (CEM) supplemented with insulin (5 ng/ml), transferrin (5 micrograms/ml), and (5 ng/ml)]. The TWI cells assume normal morphology during growth, whereas the HT-29 cells become rounded and tend to aggregate in C-ITS medium. Under these conditions the two cell types synthesize a number of different cellular proteins, and TWI releases a number of proteins to the medium. These proteins include some factors which are needed for spreading and adhesion of the cells to the growth substrate as shown by the growth of HT-29 cells in serum-free C-ITS medium conditioned by TWI cells. HT-29 cells do not synthesize such factors or perhaps, do so in negligible amounts. The serum-free medium conditioned by the TWI cells may provide an approach for the cultivation of various human tumor cells in vitro.

Comparative Studies of Chemotherapy of Human Tumor Cells In Vitro by Tritiated Thymidine Uptake Inhibition and Soft Agar Clonogenic Assay

A rapid nonsterile short-term assay has been investigated in which percent inhibition of incorporation of the DNA precursor (3H)-thymidine is measured following exposure of tumor cells to the test drugs. To evaluate the usefulness of the short-term assay in providing a rapid reliable assessment of chemotherapeutic response, the short-term assay was compared with the soft agar clonogenic assay. Sensitivity to five anticancer drugs was compared using three human tumor cell lines (epidermoid carcinoma of the oral cavity, pancreatic carcinoma, and bladder carcinoma). The short-term assay produced results that were similar to results of the clonogenic assay in two of the three tumors tested, for three drugs (cis-platin, doxorubicin, and BCNU), but did not detect responses to two antimetabolites (5-FU and MTX) in any tumor. Further studies of this short-term assay should focus on alkylating agents and other nonantimetabolites.