Diphtheria toxin effects on human cells in tissue culture.

HeLa cells exposed to a single sublethal concentration of diphtheria toxin were found to have diminished sensitivity when subsequently reexposed to the toxin. Three cells strains exhibiting toxin resistance were developed. In the cells that had previously been exposed to toxin at 0.015 mug/ml, 50% inhibition of protein synthesis required a toxin concentration of 0.3 mug/ml, which is more than 10 times that required in normal HeLa cells. There appears to be a threshold level of diphtheria toxin action. Concentrations of toxin greater than that required for 50% inhibition of protein synthesis (0.01 mug/ml) are associated with cytotoxicity, whereas those below this concentration may not be lethal. Several established human cell lines of both normal and neoplastic origin were tested for their sensitivity to the effects of the toxin. No special sensitivity was observed with the cells of tumor origin. Fifty % inhibition of protein synthesis of HeLa cells was achieved with diphtheria toxin (0.01 mug/ml) as compared to the normal human cell lines tested (0.03 and 0.5 mug/ml) and a cell line derived from a human pancreatic adenocarcinoma (0.2 mug/ml). A human breast carcinoma cell line showed a maximum of 45% inhibition of protein synthesis. This required a diphtheria toxin concentration of 5 mug/ml. These results suggest that different human cell lines show wide variation in their sensitivity to the toxin.     

Potentiation of doxorubicin cytotoxicity by (+)-1,2-bis-(3,5-dioxopiperazinyl-1-yl) propane (ICRF-187) in human leukemic HL-60 cells

The bisdioxopiperazine propane, ICRF-187, has been reported to potentiate doxorubicin cytotoxicity in certain tumor cell lines; however, the mechanism of this interaction is not known. In order to define the mechanism of this interaction, we examined the effects of ICRF-187 on doxorubicin cytotoxicity, free radical formation, and drug accumulation in human leukemia HL-60 cells. Studies show that ICRF-187 synergistically potentiated doxorubicin cytotoxicity in HL-60 cells. This potentiation of doxorubicin cytotoxicity by ICRF-187 appeared to result from enhanced drug dependent free radical formation without effecting doxorubicin uptake in HL-60 cells.     

The synthesis and antineoplastic activity of 2'-deoxy-nucleoside-cyanoboranes in murine and human culture cells.

The guanine, inosine, adenine and cytidine deoxyriboside cyanoboranes proved to be cytotoxic and possess in vivo antineoplastic activity against murine and human single cells and cultured cells derived from solid tumor lines. The agents preferentially inhibited DNA synthesis in Tmolt3 leukemic cells. The enzyme sites of drug inhibition of two active derivatives were DNA polymerase-alpha and de novo purine synthesis at the regulatory sites, PRPP amido transferase and IMP dehydrogenase. Moderate inhibition by the agents of TDP kinase and ribonucleoside reductase activities also occurred. Kinetic studies showed that IMP dehydrogenase activity was inhibited the earliest of all of the enzymes affected by the drugs. The d(ATP) pools were also reduced by drug treatment. DNA strand scission was evident after 24 hr incubation at 100 microM of drug. The 14C-cytidine-cyanoborane drug was rapidly taken up over 6 hr and most effective uptake was shown by rapidly dividing cells. The drug was bound to DNA, RNA and protein in Tmolt3 leukemic cells.     

Effect of vitamin A on methotrexate cytotoxicity in L1210 murine leukemia cells in culture

Vitamin A (VA) protects the small intestine from methotrexate (MTX)-induced damage. However, before VA can be used as a remedy to protect cancer patients from MTX-induced damage to the intestine, it is essential to clarify whether or not it disturbs the antitumor activity of MTX. This study investigated the effect of VA on the antitumor activity of MTX in vitro in L 1210 murine leukemia cells. The incorporation of [6-³H]-thymidine and [6-³H]-uridine, [5-³H]-uridine, and [4,5-³H]-leucine into DNA, RNA, and proteins, respectively, was examined to evaluate this effect. The incorporation of thymidine, the uridines, and leucine decreased dose-dependently in MTX-treated L1210 cells and profoundly in the MTX plus VA-treated L1210 cells, since VA itself had a cell-killing activity. Thus, MTX depressed the growth of L1210 cells dose-dependently and this depression was not affected by the presence of VA. The present study proved in L1210 murine leukemia cells in vitro that VA did not disturb the antitumor activity of MTX.This work was supported by a Grant-in-Aid (0367 1067) for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Prolactin receptors in human breast cancer cells in long-term tissue culture.

Prolactin receptors have been identified for the first time in a number of human breast cancer cell lines and a normal human breast cell line maintained in long-term tissue culture. Optimal conditions for determining the binding of 125I-labeled human prolactin to these cells were established. Five different tumor cell lines have different content of prolactin receptors ranging from 2,300 to 26,000 sites/cell. All tumor cell lines contained more prolactin receptors than does one normal breast cell line (1700 sites/cell). The prolactin receptors in these human mammary tumor cells not only bind human prolactin but also recognize other lactogenic hormones such as human growth hormone, human placental lactogen, and sheep prolactin, but not animal growth hormone, which are not lactogenic. The affinity (Ka) of binding of human prolactin to these cells is 4 x 10(9) M-1 (Kd = 2.5 x 10(-10)M). The hormone specificity and affinity for hormone of these human mammary tumor cells are very similar to that found for the rabbit mammary gland. These human mammary tumor cell lines in long-term culture should prove very useful to study the biology of prolactin receptors in living human cells and the role of prolactin in the tumorigenesis of the human breast.     

Prolonged retention of estradiol by human breast cancer cells in tissue culture.

Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [3H]estradiol was initially 18 nM, and after 78 hr of wash, it was 2.8 nM. After 48 hr of wash, nearly all specifically bound [3H]estradiol was present in the nucleus. Following incubation of the cells with 5 nM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed throughout the 78 hr monitored. When 10(-6) M tamoxifen or 10(-7) M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [3H]estradiol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific, and specific [3H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one-tenth of the [3H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsive cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem.     

Effect of human leukocyte interferon on the growth of human osteosarcoma cells in tissue culture.

Nine osteosarcoma cell lines, originally developed from six osteosarcoma tumours in five patients, and two cell lines of non-tumour origin (glia and fibroblast) were grown in vitro in the presence of human leukocyte interferon (L-IF). L-IF exerted a dose-dependent inhibition of growth in all these lines. The inhibitory activity displayed characteristics typical of interferons. Inhibition of cell growth occurred at a much lower L-IF concentration for the osteosarcoma than for the non-tumour-derived lines. Inhibition of tumour cell growth was observed at concentrations obtained in the serum of osteosarcoma patients treated with interferon.     

The biology of human cells in tissue culture. I. Characterization of cells derived from osteogenic sarcomas.

We have extensively characterized cell lines derived from six human osteosarcomas. The growth properties of these cultures were compared to those of fibroblastic cultures derived from skin of osteosarcoma patients and skin of bone-marrow of normal individuals. Each tumor-derived line showed some but not all of the abnormal growth properties usually associated with malignant transformation, indicating that tumor cells rather than normal stromal cells had in fact been cultured. The parameters measured included saturation density, cell morphology, growth pattern, growth on contact inhibited monolayers of normal fibroblastic or epithelial cells and tumorigenicity in immunosuppressed mice. Although the skin fibroblasts from the osteosarcoma patients appeared normal in vitro, they showed a greater ability to grow in immunosuppressed mice than did normalfibroblasts. This observation suggests that all fibroblasts of osteosarcoma patients may have an increased propensity for malignant transformation.     

Potentiation of arsenic trioxide cytotoxicity by Parthenolide and buthionine sulfoximine in murine and human leukemic cells

Purpose To possibly increase the in vitro cytotoxic activity of arsenic trioxide (ATO) by combining it with Parthenolide (PRT), a known NF-κB inhibitor and buthionine sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthetase. Methods Several cell lines representing various hematological malignancies were treated in vitro with the study drugs alone or in combinations. Flow cytometry was used to assess cell death rates and reative oxygen species production. Glutathione and ATP levels were determinded using a photometric and a luminometric assay, respectively. Cell death was characterised by fluorescence microscopy and DNA fragmentation analysis. Results PRT increased cytotoxicity of ATO in seven out of eight cell lines. Addition of buthionine sulfoximine (BSO) further potentiated cytotoxicity of the combined treatment. When combined with PRT and BSO, clinically achievable concentrations of ATO (2.5 μM) induced cytotoxicity rates of 80–98% after 24 h. Importantly, lymphocytes from healthy donors were largely unaffected by these treatment modalities, also after growth stimulation in cell culture. N-acetylcysteine inhibited the cytotoxic effects of the triple combination. Treatment of leukemic cells with ATO, PRT and BSO rapidly depleted cells from glutathione, induced oxidative stress and decreased intracellular ATP levels. Cell death showed characteristics of necrosis presumably as a result of ATP loss. Conclusion Based on the observed selectivity towards malignant cells this combination may offer a therapeutic option applicable to different kinds of leukemia.     

Enhancement by caffeine of neocarzinostatin cytotoxicity in murine leukemia L1210 cells.

Posttreatment incubation with nontoxic doses of caffeine resulted in enhancement of cell lethality and inhibition of cell growth in L1210 mouse leukemia cells which had been exposed to a protein antibiotic, neocarzinostatin. In addition, caffeine treatment appeared to inhibit the eventual maturation of newly synthesized DNA in L1210 cells following exposure to this antibiotic. These results, indicating the existence of caffeine-sensitive repair in L1210 leukemia cells treated with neocarzinostatin, provide further evidence for DNA damage as a mechanism of the cytocidal action of the antibiotic.     

Metabolism of the carcinogen 1,2-dimethylhydrazine by isolated human colon microsomes and human colon tumor cells in culture

Human colon microsomes catalyze the metabolism of the model colon carcinogen 1,2-dimethylhydrazine. Activity appears to be distributed in a gradient towards the lower end of the colon. Highest activities were observed for microsomes prepared from the descending segment of the colon with the transverse segment exhibiting lower activities, while the ascending segment showed the lowest rate of metabolism. Dimethylhydrazine metabolism in each segment is inhibited significantly by inhibitors of the cytochrome P-450-dependent mixed function oxidase system. Microsomes prepared from a human colon tumor cell also catalyze the metabolism of 1,2-dimethylhydrazine. Metabolic activity in the cell line can be induced two-fold by treatment of cells with phenobarbital and three-fold by treatment of the cells with phenobarbital plus hydrocortisone. These results show that human colon activates 1,2-dimethylhydrazine and suggest that the human colon may be capable of activating other carcinogens in situ.     

Survivin antisense oligonucleotides effectively radiosensitize colorectal cancer cells in both tissue culture and murine xenograft models

PURPOSE: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. METHODS AND MATERIALS: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, gammaH2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. RESULTS: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G(2)/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. CONCLUSION: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.     

The sensitivities of human and murine hemopoietic cells exposed to cytotoxic drugs in an in vivo culture system.

An agar diffusion chamber technique has been used to measure the sensitivities of human and murine hemopoietic colony-forming cells to cytotoxic drugs. The cells were held in i.p. diffusion chambers and exposed to the cytotoxic drugs by i.v. injection of the host mice. This method allows some account to be taken of the continuous changes in activity during the metabolic degradation of the drug. To determine how far this system provides a valid measure of the sensitivity of the cells in hemopoietic tissue, the responses of mouse bone marrow exposed to the drugs in situ in the donor mouse were compared with those of mouse cells exposed in diffusion chambers. The dose-response curves for cyclophosphamide and 5-fluorouracil were exponential in all cases. Exponential survival curves were also seen when human and mouse colony-forming cells were exposed to vinblastine or methotrexate in diffusion chambers. The plateaus seen when mouse cells were exposed to these drugs in situ could, however, be regained by omitting agar from the chambers during the exposure period. The results indicate that there are differences between the sensitivities of human and mouse marrow cells to cytotoxic drugs and that any extrapolation from mouse to humans must be viewed with caution.