The microtitre succinate dehydrogenase inhibition test for chemosensitivity of human tumour cells.
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Drug Screening Assays, Antitumor↗
Biomedical subjects
Y Sakaguchi
Publications and source records attributed to Y Sakaguchi.
Estrogen-receptor-negative breast cancer tissue is chemosensitive in vitro compared with estrogen-receptor-positive tissue.
The chemosensitivities of 18 estrogen-receptor-positive (ER+) tissues were compared with that of 38 estrogen-receptor-negative (ER-) tissues, using the in vitro succinate dehydrogenase inhibition test. These human breast tissue were exposed to six antitumor drugs: carboquone, adriamycin, mitomycin C, aclacinomycin A, cisplatin and 5-fluorouracil. Decrease in succinate dehydrogenase activity was noted in ER- compared to ER+ tissues, exposed to six antitumor drugs, in particular to adriamycin (p less than 0.001) and aclacinomycin A (p less than 0.05). The sensitive rates were higher in ER- than in ER+ tissues, against all six antitumor drugs. The resistance rates to all drugs tested were 25% in ER- and 45% in ER+ tissues. A higher chemosensitivity is associated with the absence of ER. It appeared that the ER status in case of breast cancer is an important predictor of the response to chemotherapy.
Aging↗
Combined treatment of adriamycin and dipyridamole inhibits lung metastasis of B16 melanoma cells in mice.
The combined effects of adriamycin (ADM) and dipyridamole (DP) on lung metastasis of B16 melanoma cells in mice were investigated. First, the antitumor effects of ADM and DP were examined both in vitro and in vivo. The clonogenicity of B16 melanoma cells was suppressed by the combination of ADM and the nontoxic dose of DP in vitro, and growth of the B16 melanoma solid tumor implanted subcutaneously in mice was inhibited with this drug combination, compared to findings with ADM alone in vivo. To examine events related to experimental lung metastasis, 2 X 10(5) B16 melanoma cells were given intravenously into the tail vein of mice and the efficacy was determined by counting the number of metastatic tumor nodules. DP in doses of 25-100 mg/kg given alone reduced the number of metastasis to about 85% that in the control group. ADM in doses of 1-4 mg/kg for 3 days inhibited the metastasis, in a dose-dependent manner. When combined with DP, the antimetastatic effect of ADM was enhanced and the number of metastasis prominently decreased, compared to single application of the drug (p less than 0.05). We speculate that ADM given concomitantly with DP may have the potential to inhibit metastatic growth of a tumor.
Animals↗
Detection of DNA strand breaks in HeLa cells in vitro and in mouse sarcoma 180 cells in vivo induced by an alkylating agent, carboquone, using in situ nick translation.
We determined the correlation between DNA strand breaks and the toxicity of carboquone (CQ) in HeLa cells in vitro and in mouse sarcoma 180 (S-180) cells in vivo, using in situ nick translation. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [3-H]-labelled dTTP, and sites in the DNA were visualized by autoradiographic observation of grains in the nuclei. These breaks appeared as early as 5 min in the CQ-treated HeLa cells and increased in a dose- and time-dependent manner compared to findings in the control cells, i.e., 10.2-fold at 3 x 10(-6) M in 60 min. Strand breaks in the S-180 cells appeared in a dose- and time-dependent manner, i.e., 6.1-fold after the mice had been exposed to CQ (3.6 mg/kg) for 2 h. This level correlated with the increase in host life span. Our findings show that the survival response of cells decreases, while the level of DNA strand breaks increases following exposure to CQ. The nick translation method is a rapid in situ assay for determining drug-induced DNA damage of tumor cells, under in vitro and in vivo conditions and in a semiquantitative manner.
Animals↗
[In vitro chemosensitivity test: succinate dehydrogenase inhibition (SDI) test].
The succinate dehydrogenase inhibition (SDI) test was used for determining chemosensitivity of various human tumors. This test was based on the correlation between the cellular succinate dehydrogenase activity as determined by 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT), and cell viability. The chemosensitivity varied in the tissue. Some factors are involved in the chemosensitivity, that is origin of a tumor, tissue differentiation and tissue DNA synthetic activity. This test is a convenient method for clinical use and provides important information about chemosensitivity.
Drug Screening Assays, Antitumor↗
Chicken egg white cystatin. Molecular cloning, nucleotide sequence, and tissue distribution.
A lambda gt11 chicken oviduct cDNA library was screened with a mixed synthetic oligonucleotide corresponding to amino acid residues 81-90 of chicken egg white cystatin, a cysteine proteinase inhibitor. Two initial cDNA clones of 367 and 431 bases were isolated. Both clones contained coding sequences for cystatin from amino acid residue 82 to the carboxyl end plus 3'-untranslated region and a poly(A)+ tail. The two clones utilized different polyadenylation signals located 55 nucleotides apart. Further screening of the library yielded a full-length cystatin cDNA. Sequence analysis indicated that cystatin contains an NH2-terminal extension of 23 amino acids which is probably a signal sequence. The cystatin cDNA hybridized to an mRNA of approximately 0.95 kilobase and was present in varying amounts in all chicken tissues examined. The highest concentration was found in the lung. Gizzard, brain, and heart contained lesser amounts of cystatin mRNA but considerably higher than oviduct. Among a limited number of embryonic tissues examined, significantly higher levels of the mRNA were found in liver and heart tissues when compared with the corresponding adult tissues. These results suggested that the expression of the chicken cystatin gene is tissue-dependent and under developmental control.
Amino Acid Sequence↗
5-Fluorouracil and UFT-sensitive gastric carcinoma has a high level of thymidylate synthase.
The authors examined the relationship between the level of thymidylate synthase (TS) and the sensitivity to 5-fluorouracil (5-FU) and UFT, a combined oral preparation of 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) and uracil in a molar ratio of 1:4. For the studies we used the subrenal capsule (SRC) assay and 15 human gastric cancer tissues. The TS levels were assayed by the ligand-binding technique, using [6-3H]FdUMP. The relative variation of tumor size (delta TuS/TuS0) was calculated to be as follows: delta TuS/TuS0 = [(TuS6 - TuS0)/TuS0] x 100 (%), where TuS6 was the tumor size on day 6 and TuS0 on day 0. The chemosensitivity was considered to be positive when delta TuS/TuS0 in the treated group decreased to below -10%. Decrease in tumor size was marked in case of exposure to UFT (-19.8 +/- 13.0%) (mean +/- standard deviation), compared with that to 5-FU (-9.0 +/- 7.2%), with a statistically significant difference (P less than 0.001). The TS level varied from 1.7 to 30.8 pmol/g gastric cancer tissue and the mean was 7.1 +/- 7.2 pmol/g tissue. A correlation was noted between the TS level and decrease in size of the tumor exposed to 5-FU (r = -0.671) or UFT (r = -0.758): gastric cancer tissue with higher level of TS is more sensitive to 5-FU and UFT than is that with a lower TS level. These findings show that the sensitivity to 5-FU and UFT of gastric cancer tissue is related to the TS level and that UFT shows promise for the treatment of patients with gastric cancer.
Animals↗
Effect of glucocorticoid replacement on tumor growth after adrenalectomy in mice.
We studied the effects of glucocorticoid replacement on tumor growth after adrenalectomy of Meth A sarcoma in mice. Tumor growth was inhibited in the adrenalectomized mice when a minimum dose of corticosterone, 0.3 mg/day, was given for replacement, and higher doses led to an even greater inhibition. Corticosterone had no effect on tumor growth in the irradiated mice. Sinecomitant immunity in the case of growth of the retransplanted excised tumor was compromised in the adrenalectomized mice. In vivo neutralization and immunosuppressive activities were absent in the spleen cells of the adrenalectomized mice. It would thus appear that adrenalectomy suppresses tumor growth by mechanisms other than glucocorticoid ablation. For optimum tumor control, glucocorticoid replacement after adrenalectomy should be in excess of the minimum daily requirements.
Adrenalectomy↗
Pyrimidine nucleotide synthesis is more extensive in poorly differentiated than in well-differentiated human gastric carcinoma.
In tissues obtained from patients undergoing gastrectomy, the activities of 12 enzymes involved in pyrimidine nucleotide synthesis: cytidine triphosphate (CTP) synthetase, deoxycytidine monophosphate (dCMP) deaminase, thymidine monophosphate (dTMP) kinase, uridine (Urd), deoxycytidine (dCyd) and thymidine (dThd) kinases, Urd, deoxyuridine (dUrd) and dThd phosphorylases, cytidine (Cyd) and dCyd deaminases, and DNA polymerase were examined in the eight-well-differentiated and 12 poorly differentiated gastric cancer tissues and the ten normal tissues. These cases were clinically advanced and serosal invasions were evident. Activities of these enzymes were higher in the poorly differentiated tissues than the well differentiated type and in the normal tissues. Significant differences were noted between the poorly differentiated and well-differentiated types, in dTMP kinase (P less than 0.02), dThd kinase (P less than 0.05), dThd phosphorylase (P less than 0.01), and DNA polymerase (P less than 0.05). The authors' findings show that the level of pyrimidine nucleotide synthesis, in both de novo and salvage pathways, is higher in the poorly differentiated gastric cancer tissues than in the well-differentiated type and suggest that antitumor drugs have an increased susceptibility in cases of poorly differentiated gastric carcinoma.
Adenocarcinoma↗
Sarcoma-180 cells are more sensitive to heat than are mouse normal tissues: esophagus, stomach, small intestine, large intestine, liver, spleen, and kidney.
Sensitivity of various mouse tissues to heat was determined using mouse sarcoma-180 (S-180) cells and normal tissues: esophagus, stomach, small intestine, large intestine, liver, spleen, and kidney. The in vitro succinate dehydrogenase inhibition (SDI) test was used. The succinate dehydrogenase (SD) activity of tissue fragments was assayed, following exposure to a temperature of 43 degrees C (heat treatment) or 37 degrees C (control) for 1, 2, 5, or 10 hr. The sensitivity to heat treatment was estimated by the percentage of SD activity of the heat-treated cells, compared to that of the control cells. The decrease in SD activity following exposure to heat varied with the tissue. The SD activity decreased to a greater extent in the S-180 cells than in the normal tissues. In the normal tissues, the order of sensitivity to heat was stomach, spleen, large intestine, small intestine, esophagus, kidney and liver. These results show that hyperthermia is tissue selective, hence heat treatment of a malignant lesion should be carefully designed.
Animals↗
Azo-dye-induced primary hepatoma and a gradual increase in thermosensitivity.
To determine whether or not the cytotoxic effects of hyperthermia are directed primarily to malignant cells, we examined changes in thermosensitivity during hepatocarcinogenesis in rats, as induced by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). The findings were compared with those in livers of rats fed a commercial diet. The cell viability was determined using the succinate dehydrogenase inhibition (SDI) test. The succinate dehydrogenase (SD) activity of liver cells, when exposed to heat (43 degrees C) for 2, 5, or 10 hr, decreased in a time-dependent manner, in each tissue. The decrease in SD activity was evident in 3'-Me-DAB liver for 5 hr of heat treatment on day 57, compared with findings in the normal liver. Significant differences were present for 2, 5, and 10 hr on days 93 and 136. Thus a chemically induced hepatoma is more sensitive to heat than are the normal cells. As this thermosensitivity gradually increased during the hepatocarcinogenesis, the malignant cells are particularly vulnerable to hyperthermia.
Animals↗
Species differences in substrate specificity of pyrimidine nucleoside phosphorylase.
To compare the activity of pyrimidine nucleoside phosphorylase (PNP), an enzyme involved in the metabolism of 5-fluorouracil (5-FU), we used uridine (Urd), deoxyuridine (dUrd), and thymidine (dThd) as substrates and human, rat, and mouse neoplastic and normal tissues. As PNP activity was higher in the tumor tissues than in the normal ones in all species examined, the level of PNP activity is expected to be one critical factor linked to the effectiveness of 5-FU. In rats and mice, the ratio of the activities of Urd, dUrd, and dThd was about 10:7:1, whereas in humans, the ratio was 1:30:20. The main enzyme of PNP is Urd phosphorylase in rodents and dThd phosphorylase in humans. Therefore, when examining the metabolism of 5-FU and its analogues for potential clinical application, human tissues should be used.
Animals↗
Adriamycin-induced DNA strand breaks in HeLa and in P388 leukaemia cells detected using in situ nick translation.
DNA strand breaks produced by adriamycin (ADR) were measured in HeLa cells and ADR-sensitive and -resistant P388 leukaemia cells, using the in situ nick translation method. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and 3H-labelled dTTP, and were visualized by autoradiographic observation of the grains. The DNA strand breaks in the HeLa cells increased in a dose-dependent manner, compared with findings in the untreated control cells, i.e., 15.2 fold at 20 micrograms/ml of ADR for 1 h. This level correlated with DNA single-strand breaks detected by the alkaline elution method. DNA breaks were also noted in the ADR-sensitive P388 cells, but in the ADR-resistant cells the level of DNA strand breaks was low. The enhanced cytotoxicity is apparently the consequence of the enhanced potential of ADR to cause breaks in the DNA strands. Our findings show that the survival response of the cells decreases and the level of DNA strand breaks increases following exposure to ADR. ADR resistance may be mediated by a reduction in the level of DNA strand breaks.
Animals↗
Hyperthermia enhances the in vitro activity of 1-hexylcarbamoyl-5-fluorouracil compared to that of 5-fluorouracil.
The synergic effects on HeLa cells between 1-hexylcarbamoyl-5-fluorouracil (HCFU), a lipophilic masked compound of 5-fluorouracil (5-FU), and hyperthermia were investigated, with the following results: (1) After the cells had been exposed to the drug at 77 microM for 1, 2 or 3 days, with or without heat (43 degrees C) for 2 h, treatment with HCFU and heat inhibited cell growth by 9.2% on day 3, compared to control cells, HCFU alone, heat alone or the combined treatment with 5-FU plus heat. (2) The cells were exposed to the drugs at 77 microM or heat (43 degrees C) for 15-45 min and the colonies counted on day 14. Combined treatment with HCFU plus heat inhibited the clonogenicity of HeLa cells to 22.4% of findings in the control cells and as compared to findings with other treatments. The combination of 5-FU, n-hexylamine and heat had much the same effect as the combined effect of the treatment of HCFU plus heat. (3) Intracellular levels of the drugs did not increase with hyperthermic treatment. Our findings suggest that it is the hexylcarbamoyl structure of HCFU that relates to the synergic effect between HCFU and hyperthermia. This combination is expected to have positive effects for treating patients with malignancy.
Antineoplastic Agents↗
Improvement in the surgical results of treatment of duplicated thumb by preoperative splinting.
Seven patients with an angular deformity of their duplicated thumb were treated by splinting and taping before operation. In all patients, the angular deformity of the interphalangeal joint improved within a few months, and surgical centralization and osteotomy were not necessary. Furthermore the design of the fillet flap was easier and more accurate as was the adjustment of the joint ligament tension. After operation there was no recurrence of angular deformity, and the range of motion of the interphalangeal joint was good. We believe that this procedure simplifies the operative approach, making it more precise and helping to prevent postoperative complications in patients with angular deformity.
Hand Deformities, Congenital↗
In vitro and in vivo antibacterial activities of AT-4140, a new broad-spectrum quinolone.
AT-4140, 5-amino-1-cyclopropyl-6,8-difluoro-1,4-dihydro-7-(cis-3,5- dimethyl-1-piperazinyl)-4-oxoquinoline-3-carboxylic acid, showed broad and potent antibacterial activity. Its MICs for 90% of the strains tested were 0.1 to 0.78 micrograms/ml against gram-positive organisms, such as members of the genera Staphylococcus, Streptococcus, and Enterococcus, and 0.0125 to 1.56 micrograms/ml against gram-negative organisms, such as members of the family Enterobacteriaceae and the genera Pseudomonas, Branhamella, Campylobacter, Haemophilus, and Neisseria. Its MICs were 0.025 to 0.78 micrograms/ml against glucose nonfermenters, such as members of the genera Xanthomonas, Acinetobacter, Alcaligenes, Moraxella, Flavobacterium, and Brucella; 0.2 to 0.78 micrograms/ml against anaerobes, such as Clostridium perfringens and Bacteroides fragilis; 0.0125 to 0.05 micrograms/ml against Legionella spp.; 0.0125 to 0.2 micrograms/ml against Mycoplasma spp.; 0.031 to 0.063 micrograms/ml against Chlamydia spp.; and 0.1 to 0.3 micrograms/ml against Mycobacterium spp. The potencies of AT-4140 against gram-negative organisms were comparable to those of ciprofloxacin and higher than those of ofloxacin, enoxacin, and norfloxacin. The potencies of AT-4140 against gram-positive organisms, glucose nonfermenters, anaerobes, Mycoplasma spp., Chlamydia spp., and Mycobacterium spp. were generally higher than those of the quinolones with which AT-4140 was compared. AT-4140 showed good oral efficacy against systemic infections with Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, and Pseudomonas aeruginosa in mice. Its efficacy was better when a daily dose was given once than when it was given in two doses. Good efficacies of the orally administered drug were also observed in pulmonary, dermal, and urinary tract infection models in mice. The in vivo efficacies of AT-4140 were equal to or better than those of ciprofloxacin, ofloxacin, enoxacin, and norfloxacin.
Animals↗
Hypoxia enhances the lethality of mitomycin C and carboquone against human malignant tumor cells in vitro.
Solid tumors contain hypoxic cells which are relatively resistant to antineoplastic activity of radiation or to chemotherapeutic agents. We determined the increase in antineoplastic activities of mitomycin C (MMC) and carboquone (CQ) against human tumor cells under conditions of in vitro hypoxia, with the following results. (a) The HeLa cells had been exposed to various concentrations of MMC or CQ for 2 h under normally aerated (about 20%) or hypoxic conditions (5.0 and 0%) and were maintained under normally aerated conditions for 3 days. The succinate dehydrogenase activity was assayed, using the succinate dehydrogenase inhibition (SDI) test. Treatment with MMC or CQ reduced the SD activity, compared to findings in the control cells, under hypoxic conditions. (b) The cells were exposed to the drug, MMC or CQ, under normally aerated or anoxic conditions, for 15-45 min and the colonies counted on day 14. Treatment with 0.45 microM of MMC for 45 min inhibited the clonogenicity of HeLa cells by 25.4% of findings in the control cells under anoxia. Treatment with 0.03 microM of CQ for 30 min inhibited the clonogenicity by 41.5%. (c) The sensitivity of 14 human tumor tissues (6 gastric and 8 colorectal cancers) to MMC or CQ under hypoxic conditions was determined, using the SDI test. MMC and CQ showed hypoxic enhancement. Therefore, MMC and CQ have definite positive effects on hypoxic cells, and hence a higher therapeutic ratio for treating clinical solid tumors.
Azirines↗
[Detection of cellular DNA strand breaks induced by antitumor drug and heat using in situ nick translation].
Cellular DNA strand break induced by an alkylating agent: Carboquone (CQ), and heat (43 degrees C) was detected in HeLa cells in vitro and mouse sarcoma-180 cells in vivo. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [3H]-labeled dTTP and sites in the DNA were visualized by autoradiographic observation of grains in the nuclei. These breaks increased in a dose and time dependent manner, compared to findings in the control cells. Our findings show that the surviving response of cells decreases while the level of DNA strand breaks increases following exposure to CQ or heat. The nick translation method is a rapid in situ assay for determining drug and heat induced DNA damage of tumor cells, under in vitro and in vivo conditions and in a semi-quantitative manner.
Azirines↗