Tumor necrosis factor (TNF) is cytokine derived from macrophage and shows much promise for application in cancer therapy because of its marked antitumor effects and its high specificity to tumors. Recently, the gene encoding human TNF was expressed in E. coli and the recombinant TNF was purified to homogeneity by ion exchange chromatography, affinity chromatography, and gel filtration. Clinical study of rH-TNF has been launched because human recombinant TNF can be produced on a large scale. In spite of notable antitumor effects, little is known concerning the mechanism of action of cytotoxic activity. In this article, the mechanism of action of rH-TNF against tumors in vitro and in vivo is reviewed.

Although the human tumor clonogenic assay (HTCA) is extremely reliable in determining clinical correlations, it is a complicated process requiring considerable time in order to obtain results. Thus, an experimental study on cytopathologic observation (cytologic assay) and comparative evaluation between it and HTCA were performed in order to establish a more rapid and accurate drug sensitivity test. Materials included Colon 26, a cell line established in our department, malignant effusion and surgical specimens. In carrying out HTCA according to the Hamburger-Salmon method, the cell suspension samples following exposure to anti-tumor agents (MMC, L-PAM, ADM, CDDP) were cultivated in test tubes for 3-8 hours and stained by the Papanicolaou and Giemsa methods. According to Tokita's criteria, when cellular changes showed as nuclear pyknosis and nuclear destruction were found to have increased significantly in comparison with a control group, the cells were judged to be sensitive. Very similar and parallel results were obtained between HTCA and cytologic assay in this study, with a significant correlation. Cytologic assay was proved to be an easy, rapid and accurate method for testing drug sensitivity and its clinical application can be expected in the future.

Subrenal Capsule Assay (SRCA) as a chemosensitivity test was performed on 14 esophageal squamous cell carcinomas in order to select a more effective form of chemotherapy. Of the 14 assays, 12 were evaluable. Mice were treated with anticancer agents (e.g. Cisplatin, Bleomycin, Methotrexate, Vindesine) on days 1 and 3 after transplantation, and on day 6, the sensitivities were determined. Fresh esophageal cancers yielded an evaluable assay rate of 74%. The implant grew progressively for six days in the remaining group of control mice. Histologically, host cell infiltration at the border of the implant was observed from day 3 after transplantation, and cells had degenerated or had been partially replaced by scar tissue by day 6. The results of chemosensitivity tests differed according to the anticancer agent used or from case to case. Clinically, correspondence between the assay results and clinical results was obtained in 5 out of 7 cases. SRCA is a new promising chemosensitivity test which is clinically useful, and the present results indicated the feasibility of its use in developing an effective chemotherapy for esophageal cancer.

The predicted level of cell viability was compared between the succinate dehydrogenase inhibition (SDI) test and the adenosine triphosphate (ATP) assay, both of which are used for in vitro human tumor chemosensitivity testing. After HeLa cells had been exposed to various concentrations of 5-FU for 1, 2, 3 or 4 days, the decrease occurring in viable cell number correlated with that of succinate dehydrogenase (EC 1.3.99.1) activity and that of the intracellular ATP level of the viable cells. In dead cells, the ATP level was extensively decreased, but the succinate dehydrogenase activity remained at a level of 11% of that of 5-FU-untreated viable cells, even on day 4. The cell viability correlated well with the intracellular ATP level, as compared with the succinate dehydrogenase activity. The activity remaining in dead cells must thus be taken into consideration for the prediction of chemosensitivity in the SDI test, but not in the ATP assay.

Sister chromatid exchanges (SCEs) induced by four anticancer drugs, 3-(4-amino-2-methyl-5-pyrimidyl) methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU), 1-3-bis (2-chloroethyl)-1-nitrosourea (BCNU), nitrogen mustard (HN2), cis-diamminedichloroplatinum (II) (cis-Pt) were examined on five cell lines derived from human malignant glioma biopsy specimens, and compared to results obtained with colony-forming efficiency (CFE) assay. Treatment of the five cell lines with these four drugs produced concentration-dependent increases in SCEs. Treatment with ACNU induced the most SCEs in SF-126 cells decreasing in SF-268 cells followed by SF-210 cells, SF-295 cells, and the least SCEs in SF-188 cells. The results of the SCE assay with BCNU in these cell lines were similar with ones with ACNU. In contrast to results obtained with nitrosoureas, the most SCEs were induced in SF-188 cells and the least were induced in SF-126 cells by the treatment of HN2. The frequency of SCEs induced with cis-Pt was almost similar in the five cell lines. The number of SCEs induced by the treatment of ACNU, BCNU, HN2 and cis-Pt in five cells lines showed a good correlation with cytotoxicity measured by CFE assays, and induction of SCEs occurred at much lower concentrations of these anticancer drugs than those required to induced cell kill. These results suggest that measurement of induced SCEs in human brain tumor cells treated with some anticancer drugs provide a more sensitive indicator of drug action than CFE assay and that SCE assays may be a useful method of the in vitro sensitivity test to some anticancer drugs.

Experiments were performed to ascertain whether or not the cytotoxic effects of various anticancer drugs on five human genitourinary malignant cell lines would be enhanced by recombinant gamma-type interferon. The cells used were as follows: HeLa cells from a uterine cervix cancer, HT-1376 and EJ cells from bladder cancers, ACHN cells from a renal cancer, and PC-3 cells from a prostatic cancer. The effects of the drugs were studied by colony formation assay. The following drugs were used: two metabolic antagonists. cytosine arabinoside (Ara-C) and 5-fluorouracil (5-FU), three antibiotics: adriamycin (ADM), mitomycin C (MMC) and peplomycin (PEP), two alkylating agents: nimustine hydrochloride (ACNU) and melphalan, one vinca alkaloid: vincristine (VCR) and one other drug: cisplatin (CDDP). Interferon used was a preparation of recombinant gamma-type interferon. PEP showed synergistically enhanced cytotoxic effects on HeLa, EJ, HT-1376, and ACHN cells by concomitant application with gamma-IFN. Synergistic cytotoxicity was also detected against HeLa, EJ and ACHN by combined treatment with ADM and gamma-IFN. A similar enhanced cytotoxicity was demonstrated in HT-1376 and PC-3 by 5-FU treatment with gamma-IFN. MMC showed enhanced cytotoxicity only against ACHN cells in the presence of gamma-IFN. The cytotoxic effects of PEP on cells were increased by lower concentrations of gamma-IFN compared with those of other drugs. DNA, RNA and protein synthesis were examined in HeLa cells following combined exposure to PEP and gamma-IFN. The combined therapy was found to produce a specific decrease in DNA synthesis, while yielding no significant inhibition of intracellular RNA and protein synthesis.

An adriamycin-resistant cell line was established in culture by continuous exposure of SBC-3 cells, a cell line of human small-cell lung cancer, to increasing concentrations of adriamycin, followed by a cloning procedure. The resistant cells (SBC-3/ADM) were 30 times more resistant to the drug than the parent cells in terms of 70% lethal dose, determined by soft agar clonogenic assay. Uptake studies using [3H] daunomycin, which was completely cross-resistant to adriamycin, showed decreased influx and enhanced efflux of the agent. This resistance to adriamycin may be attributed to an alteration in membrane transport, resulting in reduced intracellular accumulation of the drug. Using the SBC-3/ADM cells, the activity of a variety of drugs was analyzed by soft agar clonogenic assay in order to search for a means of circumventing drug resistance. The SBC-3/ADM cells were markedly resistant to anthracycline antibiotics such as THP-adriamycin and 4'-epi-adriamycin. The cells were also resistant to structurally or pharmacodynamically unrelated compounds such as vincristine, mitomycin C, 40497S, an active compound of ifosfamide, and etoposide. However, the cells were as sensitive to mitoxantrone as the parent cells, and were considerably susceptible to cisplatin. These results suggest that mitoxantrone and cisplatin may exert a sufficient degree of activity for use against small-cell lung cancer resistant to adriamycin.

It is difficult to predict the adequate doses of effective drugs which must be administered in cancer chemotherapy. We have developed a successive colony-forming assay in order to analyze changes of drug sensitivity in human cancer cell lines, and it is used as a model of cancer chemotherapy. Presently, widely used methods include the clonog ceni assay as in vitro sensitivity test established by Salmon and Hamberger et al., for revealing effective drugs. However, formation of a second colony using initial colony-forming cells has not yet been performed. We therefore analyzed the changes occurring in drug sensitivity in 2nd and 3rd colony-forming assays. Using a human lung adenocarcinoma cell line PC-9 (established by Hayata at the Surgical Department in Tokyo Medical School), we measured the changes in sensitivity to CDDP and its derivatives. After picking up the colony-forming cells by micropipette and culturing them in culture medium, we tried to incubate them for 1 hour with the anticancer drug, and then replate them in soft agar. This technique is called "2nd colony-forming assay". We were able to carry this out three times. The results obtained showed that the anti-tumor effect of 254S was the same as that of CDDP, whereas that of CBDCA was less than either of them. Although these tendencies were noticeable even in the 2nd colony-forming assay, the sensitivities to CDDP and its derivatives were diminished in the 3rd colony-forming assay.

We carried out in vitro anticancer drug sensitivity tests a total of 24 times in 15 cases of childhood leukemias and lymphomas using the microplate culture and crystal violet staining method. We then compared them with the clinical effects. As a result, the in vitro efficacy rate at onset of cases was 40%, which was less than that for relapsed cases, which was 100%. Especially, cases of ALL at onset all failed. As for the relation between in vitro and in vivo effects, cases for which it was possible to make evaluation both in vitro and in vivo numbered 16. The results for these cases were; true positive rate, 71%; true negative rate, 50%; predictive accuracy, 69%. The true positive rate was higher than the usual rate. As for the efficiency of each anticancer drug, in vitro predictive accuracy for ADR, Ara-C and 4 HCP (CPM) was higher than for other drugs. From these results, it became clear that our method is very useful for the screening of effective anticancer drugs.

We have applied the MTT dye reduction assay to the anticancer drugs sensitivity test using short-term microplate cultures. The tumor cells were cultured with the anticancer drugs for 2 and 4 days. After culture, MTT dye was placed in each microwell and culture was carried out again for 4 more hours. The formazans generated by living cells were dissolved in acidified isopropyl alcohol and the absorbances of each well were measured at a wavelength of 540 nm. When tables of cytotoxicity indices classified into anticancer drugs, concentrations and durations of culture for each type of leukemic cell were made, it became possible to compare each drug and to select the effective ones. This assay is simple, precise, rapid, has no washing steps and is convenient for handling a large volume of material. We apply this assay in clinical practice.

A 53-year-old male with thalamic degeneration is presented. He had double vision, cerebellar signs, and pyramidal and extrapyramidal tracts signs in addition to hypersomnia, decrease in spontaneity and attention, and impairment of memory as psychic symptoms. These signs and symptoms were progressive, and he subsequently developed akinetic mutism and died of pneumonia 17 months after the onset of the disease. His clinical diagnosis was considered as Gerstmann-Sträussler syndrome due to progressive dementia, cerebellar signs and the other signs mentioned above. The postmortem pathological investigations, however, revealed thalamic degeneration. The pathological observations showed marked loss of nerve cells and glial proliferation in the medial and anterior nuclei of thalamus. The same pathological changes were more or less demonstrated in the pulvinar, the periaqueductal gray matter of midbrain, inferior olivary nucleus, the medial parts of globus pallidus, the substantia nigra and the dentate nucleus. In the early stage of the clinical course, it was difficult to know whether the main symptoms were caused by dementia or by the disturbance of consciousness. Retrospective considerations, however, showed that dementia had appeared at first, and subsequently the disturbance of consciousness had joined. As the result, it seems that they finally caused akinetic mutism. It is known as thalamic dementia that in the cerebrovascular disease the lesions in the medial and anterior parts of bilateral non-specific thalamic nuclei cause dementia.(ABSTRACT TRUNCATED AT 250 WORDS)

An autopsy case of Shy-Drager syndrome preceded by urinary disturbance for over 20 years was reported. A 43-year-old woman was admitted to our hospital because of urinary disturbance and orthostatic hypotension. At the age of 19 she developed urinary disturbance with polyuria and retention. These symptoms were getting worse with years, and at the age of 33 she was diagnosed to have neurogenic bladder of uninhibited type. During her hospital course her symptom became worse, and by the age of 42 she showed marked dysarthria, disturbance of smooth pursuit eye movement, Horner's syndrome, marked rigidity and tremor of four extremities, generalized hyperreflexia, marked limb and truncal ataxia, neurogenic bladder and orthostatic hypotension. Serial brain CT scan revealed progressive brain stem and cerebellar atrophy with clinical course. Severe autonomic nervous system dysfunctions were also documented. She died of respiratory failure at the age of 43. On autopsy, brain stem and cerebellum showed marked atrophy macroscopically. Microscopically marked depletion of neuron was seen in the substantia nigra, pontine nuclei, inferior olive, Purkinje cells, the intermediolateral column of spinal cord and Onuf's nucleus of S2. Although numerous cases of Shy-Drager syndrome have been reported in the past, there is no case which developed this syndrome after urinary disturbance of over 20 year's duration. We should be alert to observe the cases with longstanding urinary disturbances in order to not overlook degenerative disorders as exemplified in this case.

The sensitivity to actinomycin-D (Act-D) and the changes in survival rate of two human choriocarcinoma cell lines (BeWo and SCH) were studied in vitro and the following results were obtained. BeWo was shown to be more sensitive to Act-D than SCH, when the survival rate was compared in the two cell lines. 3H X Act-D uptake was 39 pmol/10(6) cells in BeWo and 12 pmol/10(6) cells in SCH after a two hours treatment. Those results suggested that the sensitivity to Act-D of choriocarcinoma cells was positively correlated with the intracellular Act-D concentration. The intracellular Act-D concentration was increased depending upon the concentrations of amphotericin B (AMB). After a two hours treatment with Act-D and AMB, the intracellular Act-D concentrations were twice in BeWo, and 2.3 times in SCH comparing with those treated with Act-D alone. The synergistic effects of Act-D and AMB on the survival rate were 1,000 times in BeWo and 100 times in SCH compared with those treated with Act-D alone. From the above, combination therapy with Act-D and AMB was supposed to be one of the trial methods in the treatment of drug resistant choriocarcinoma.

After serial transplantation in nude mice, we had established three new human choriocarcinoma cell lines (NaUCC-1,2 and 3). These three cell lines and BeWo were examined for sensitivity to Act-D, MTX and the combined agents (Act-D + MTX) 3H-Act-D uptake and 3H-MTX uptake, and were compared for each treatment. NaUCC-1 showed low sensitivity to MTX (p less than 0.05), but showed high sensitivity to Act-D (p less than 0.05). BeWo showed low sensitivity to Act-D (p less than 0.05). In examining sensitivity to the combined agents (Act-D+ MTX), the sensitivity of NaUCC-2 to Act-D was decreased (p less than 0.05) by MTX added at the same time. In the 3H-Act-D uptake experiment, NaUCC-1 did uptake a relatively larger amount of 3H-Act-D (p less than 0.01) and BeWo a smaller amount of it (p less than 0.05). In the 3H-MTX uptake experiment, NaUCC-1 did uptake a relatively smaller amount of 3H-MTX (p less than 0.05), but NaUCC-2 uptook a larger amount of it (p less than 0.005). NaUCC-1 established from the patient in whom tumor cells were resistant to treatment, had a low response to MTX. NaUCC-2 was established from the patient in whom it was found that MTX inhibits the Act-D effect on tumor cells. In the study of combined agents in NaUCC-2, the growth inhibition effect of Act-D was suppressed by the MTX added.

In an effort to develop more nearly optimal conditions for the clonal growth of hemopoietic stem cells (CFC, BFU-E, CFU-mix and Mast-CFC) iv vitro, I examined the influence of low oxygen tension (7% O2) on plating efficiency. The numbers of colonies derived from human bone marrow CFC increased by 1.7 fold in 7% O2 than under a gas phase containing air (19% O2). Bursts obtained from bone marrow BFU-E and mixed colonies from CFU-mix increased by about 2.5 fold in 7% O2. Total cell count of mixed colonies showed that the average cell per colony gassed with 7% O2 were 900 compared with 511 in 19% O2. However the subpopulation of CFC and composed cell type of mixed colonies were not different in the two gas phase. With mouse spleen cell in 7% O2 a dramatic increase in Mast-CFC numbers without 2-ME and decreased enhancement by 2-ME were seen. Blood gas analysis of human bone marrow showed a Po2 of 51.8 +/- 14.5 mmHg, which was close to O2 tension in culture media of gas phase contained 7% O2. These data showed that physiological O2 tension enhances hemopoietic stem cell proliferation in vitro, and that part of the enhancing effect by 2-ME is due to a prevention of O2 toxicity at 19% O2.

A soft agar colony formation assay, so called human tumor clonogenic assay (HTC assay) similar to that originally described by Salmon and colleagues, was utilized to measure the sensitivity of a total of 85 urologic malignancies including 36 urothelial cell carcinomas, 41 renal cell carcinomas, 5 testicular tumors, and 3 Wilms' tumors to anticancer drugs. In addition, the results obtained were compared with those of a novel dye exclusion method (NDE assay) described by Weisenthal and colleagues. The NDE assay was utilized to measure the sensitivity of a total of 63 urologic malignancies including 28 urothelial cell carcinomas, 25 renal cell carcinomas, 6 testicular tumors, and 4 Wilms' tumors to anticancer agents. In both assay series, the concentration of anticancer drugs tested was approximately one tenth of the maximum serum level achievable after single bolus injection. The colony forming rate inhibition of 70% or more in the HTC assay and the cell survival rate of 30% or less in the NDE assay were defined as "sensitive." Sixteen of the 36 urothelial cell carcinomas, 11 of the 41 renal cell carcinomas, and 1 of the 5 testicular tumors had both more than 30 colonies grown in control plates and enough cells in the specimens to provide at least one drug sensitivity testing. In urothelial cell carcinomas, 3 out of 13 tumors were "sensitive" to adriamycin, 3 out of 16 to cis-platinum, and 4 out of 15 to carboquone. In renal cell carcinomas, 2 out of 9 tumors were "sensitive" to adriamycin, 4 out of 11 to vinblastine, and none of 4 to Interferon alpha.(ABSTRACT TRUNCATED AT 250 WORDS)