In order to study the growth of granulocyte-macrophage colony-forming unit (GM-CFU), fundamental studies on in vitro colony formation were carried out using mouse bone marrow cells by the soft agar culture and the following results were obtained: The number of colonies increased to a maximum level after 4 days of incubation and decreased thereafter. There was a positive correlation between the number of colonies and that of cultured bone marrow cells. There was a linear dose-response relationship between the number of colonies and the concentration of colony stimulating factor (CSF), and the maximum response was obtained at the concentration of 10% CSF. As the fetal calf serum (FCS) dose increased, the number of colonies also multiplied. No significant difference was observed in the colony forming ability between non-separated and separated (non-phagocytic) cells in bone marrow cells. The addition of 2-mercaptoethanol increased doubled the number of colonies at the concentration of 1 X 10(-4) M. The results of these fundamental studies on GM-CFU are thought to be useful for the investigation of granulopoiesis.

Fundamental studies on CFU-E colony formation were done using bone marrow cells of mice by the methylcellulose culture method. The number of CFU-E colonies reached a peak after 48 hours of incubation. A linear relationship was observed between the number of CFU-E colonies and the number of inoculated bone marrow cells. The number of CFU-E colonies increased dependently on the concentration of erythropoietin (Epo) and reached almost a plateau above 0.25 U/ml. There was little difference in the CFU-E colony forming activity of the two sources of Epo (one partially purified from human urine at our laboratory and the other obtained from Connaught Laboratories Ltd. as Step III derived from plasma of anemic sheep). There was little difference in the number of marrow CFU-E of two strains of mice (ddY and BDF1). The best conditions in the concentration of culture medium were 10-30% in FCS, 0.5-1.0% in BSA, 10(-4) M in 2-mercaptoethanol, respectively. These observations are thought to be useful for the development of studies on CFU-E.

A 10-year-old boy had gait and speech disturbances 17 days after the initial symptoms of a fever, headache and cough. Four days later he was admitted to a hospital with mild disturbances of gait, speech, writing, visual acuity, left facial nerve, nystagmus and consciousness. Impairments of cranial nerves (II, III and VII), pyramidal sign and cerebellar sign were noticed. EEG showed generalized slow waves. Auditory brain stem response showed prolongation of the interval between I and V waves and poor differentiation between them. Brain CT could not find any abnormalities. Brain stem encephalitis was diagnosed. Clinical signs and symptoms continued for two weeks when steroid therapy was started and it was effective to improve the disease. He was discharged from the hospital without sequelae. Herpes simplex virus (HSV) type 1 was detected from cells in CSF on admission by fluorescence antibody method. HSV antibody titers in sera changed from 1/8 to 1/64 during three months by complement fixation test. Specific IgG and IgA by enzyme linked immunosorbent assay (ELISA) was high in CSF. Specific antibody in CSF/total antibody in CSF: specific antibody in serum/total antibody in serum for IgG and IgA classes were more than 1. Reports of mild type of HSV brain stem encephalitis seemed to be rare. Our case which was followed for several months carefully would be important to discuss.

After microsopically-directed sampling of the tissues from various histological sections taken from 16 cases of cervical dysplasia, 6 cases of carcinoma in situ, 11 cases of stage Ib invasive cervical cancer of keratinizing type, 28 cases of large cell non-keratinizing type and 14 cases of small cell type, nuclear DNA levels of the cells dispersed from the tissues were measured by cytofluorometry after Feulgen stain. The DNA levels of cells obtained from normal cervical squamous epithelium and squamous metaplastic epithelium were in 2C(diploid)-4C(tetraploid) regions and those from mild and severe dysplasia were in 2C approximately 4C or high 4C and in low 2C or 2C approximately high 4C or 8C(octaploid) regions respectively with the mode of 2C. In five of 6 cases (83.3%) of carcinoma in situ, the amount of DNA in the neoplastic cells ranged up to the hyperoctaploid region with the mode of 2C or 4C. There were hyperoctaploid cells in 90.9% of cases of the keratinizing type, 96.4% of cases of the large cell non-keratinizing type and 100% of cases of the small cell type. The incidence of hyperoctaploid cells in samples from superficial invasion (stromal invasion less than 3mm) was not different from that of deep invasion (stromal invasion of more than 5mm) in each histological type. When the modal values for nuclear DNA in the superficially invasive lesions were compared with those of the deeply invasive lesions, aneuploidy was more frequently observed in the lesions of deep stromal invasion, irrespective of the histological type. The dominant changes in the mode according to the depth of stromal invasion were 2C to aneuploid in keratinizing type and 4C to aneuploid in cases of large cell non-keratinizing type and small cell type. The results suggest that the hyperoctaploid and aneuploid cells are useful markers for quantitative discrimination among dysplasia, carcinoma in situ and invasive squamous cell carcinoma and that aneuploid stem cells may be generated from 2C and 4C stem cell lines in the progression of stromal invasion.

Cytotoxic and cytokinetic effects of Cisplatin (cisdiamminedichloroplatinum) on cultured human and rat glioma cells (KY and C6) were studied both in vitro and in vivo. The cytotoxic or cytostatic effect was evaluated by either colony formation assay or inhibition rate of cell growth. The cytokinetic effects were analyzed by DNA histogram using a flow cytometer. It was found that the cytotoxic effect of Cisplatin on the cultured glioma cells was dose-dependent, and that human KY cells and rat C6 cells had similar sensitivity to Cisplatin. Within 24 hours after treatment of KY cells with Cisplatin, a time-dependent effect was observed, but the cytotoxic effect of Cisplatin in the medium decreased rapidly. Investigation of chronological changes in the DNA histogram of KY cells treated with 0.2 microgram/ml Cisplatin revealed that cell-cycle progression was delayed in the S-phase and blocked at the S-G2 + M boundary 18 hours later. KY tumors subcutaneously transplanted into nude mice were treated with intraperitoneal injection of 3 mg/kg/day Cisplatin for 10 days and the tumors markedly regressed by 65% of their wet weight.

On twenty-three specimens from patients with osteosarcoma (biopsied tissue, resected specimens from primary and metastatic lesions), the human tumor clonogenic assay (HTCA: standard assay) as well as cultivated HTCA involving short-term cultivation of a single-cell suspension, was carried out. The percentage colony-forming ability, which was very small for standard HTCA, increased significantly when cultivated HTCA was performed. With cultivated HTCA, sensitivity tests were possible in 87% of the specimens, in contrast to only 28.6% with standard HTCA. Retrospective evaluation of the results obtained by cultivated HTCA with the clinical efficacy of anticancer drugs showed a true positive rate, 66.7%, and a true negative rate, 88.9%. This analysis revealed that drugs shown to be ineffective and that tumor cells are refractory to those drugs, so that clinical use of such drugs should be avoided.

We attempted to study the relation between 5-FU incorporation into RNA or DNA and the cytocidal effect of 5-FU using thymidine (TdR) and the effect of N-phosphoacetyl-L-aspartate (PALA) on the cytotoxicity of 5-FU. A mouse mammary carcinoma cell line FM3A was used in all of the experiments. The results showed that: addition of TdR to cell cultures resulted in an increase of 5-FU incorporation into RNA and although 5-FU incorporation into nucleic acid decreased, addition of 10(-3) M TdR induced of complete incorporation of 5-FU into only RNA as revealed by alkaline hydrolysis and cesium gradient analysis; no addition of TdR caused 5-FU to be completely incorporated into DNA only; cytocidal effect of 5-FU, in a clonogenic assay, occurred only during the time when TdR was added to the cell culture; PALA possessed the capacity to enhance the cytocidal effect of 5-FU in a clonogenic assay. These results confirmed that 5-FU incorporation into RNA is necessary for the cytocidal effect of 5-FU and that both TdR and PALA are available as 5-FU-modulating agents for enhancement of the cytocidal effect.

The clinical results and problems of extracorporeally-induced total-body hyperthermia (TBHT) for recurrent cancer were presented. A total of 105 hyperthermic treatments were performed in 38 patients who had had unsuccessful conventional systemic anticancer chemotherapy. Partial response was observed in 10 of 29 evaluable patients (37%). In analysing the anticancer effects of TBHT according to cancer site, a high efficacy was observed in patients with their main tumor in the lung, liver and lymph nodes. The anticancer effects were most enhanced when TBHT was performed in combination with cis-diamminedichloroplatinum (II) and 5-fluorouracil. In order to augment the anticancer effects of TBHT, the choice of combined agent(s) and administration timing are important. A useful method for determining the thermochemosensitivity of individual cancer cells to agents selected for drug treatment is the human tumor stem cell assay. Further, the usefulness of angiotensin II-induced hypertensive chemotherapy during TBHT for augmenting selective drug delivery to cancer tissues is stressed.

To improve the plating efficiency (PE) of cells used in the standard human tumor clonogenic assay (standard HTCA), an HTCA was developed using single-cell suspensions obtained from short-term cultures of surgical specimens of malignant bone and soft tissue tumors of the extremities. Consequently, the percentage possibility of application for the chemosensitivity test for anticancer drugs as well as the PE improved from 29.7% to 65.7% for bone tumors (37 specimens) and from 30.0% to 72.4% for soft tissue tumors (31 specimens). In bone tumors the correlation between these drug sensitivity tests and the clinical effectiveness of the drugs was retrospectively evaluated in 17 specimens from 13 cases mainly of osteosarcoma; they were well correlated with the true positive rate (71.0%) and the true negative rate (90%). In soft tissue sarcomas, retrospective evaluations were possible only in 9 cases and their true positive and negative rated were 75.0% and 100% respectively. These results indicate that cultivated HTCA seemed to be a clinically useful chemosensitivity test: the anticancer drugs judged useless by this test were likely to be clinically useless. Drugs such as resistance-acquired or non-sensitive drugs should be excluded from multidrug combination chemotherapy in the treatment of bone and soft tissue sarcoma.

In order to determine the optimal conditions of the clonogenic assay for predicting antitumor activity in vivo, experimental chemosensitivity tests in vitro and in vivo were performed using a human gastric carcinoma strain, H-111 serially transplanted into nude mice. According to the method of Salmon and Hamburger, 5-fluorouracil (5-FU) was added to dissociated tumor cells at concentrations of 0.1, 1 and 10 micrograms/ml continuously for 2 weeks in the upper layer of a double agar preparation. The transplanted H-111 tumors in nude mice were treated with 30, 60 and 120 mg of 5-FU per kg. The antitumor activities were evaluated by the T/C ratio of the colonies in vitro and the T/C ratio of tumor weights in vivo. Pharmacokinetic analysis of 5-FU was conducted by bioassay with Staphylococcus aureus in the agar and in the serum of mice. When areas under the curves in vitro and in vivo were supposed to be equivalent, the antitumor activities of 5-FU in vitro and in vivo could be correlated quantitatively in terms of T/C ratios. The drug concentration in the clonogenic assay for predicting the effect of the MTD of 5-FU in vivo (50 mg/kg X 3) was calculated to be 1 microgram/ml for 2 weeks.

The authors reviewed their experiences to date with chemosensitivity testing of 629 tumors by human tumor clonogenic assay (HTCA) and of 199 tumors by scintillation assay (SA). HTCA and SA were both performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Overall, 60% of specimens in HTCA and 58% in SA produced significant growth in vitro. HTCA was 52% (13/25) reliable for predicting in vivo sensitivity, and 95% (36/38) reliable for in vivo resistance, whereas SA was 40% (8/20) reliable for in vivo sensitivity and 88% (21/24) for in vivo resistance. The current results indicate that there is no particular difference in the success rate and in the predictive accuracy for in vivo sensitivity and resistance between HTCA and SA. Therefore, it is doubtful whether only clonogenic tumor cells among whole tumor cell populations should be selected in assessing the chemosensitivities of human tumors. In vitro success rates were variable, depending on the tumor histology. In vitro growth of gastric cancer specimens was characteristically lower than that of colon cancer specimens (48% and 60% in HTCA, and 46% and 68% in SA, respectively). (p less than 0.005). Optimal in vitro-in vivo drug concentrations and culture conditions are still being defined. Correlation studies of in vitro-in vivo responses of gastrointestinal cancers suggested that in vitro concentrations of 5-fluorouracil and mitomycin C used in this study were considerably higher than their optimal doses. Tumor cell heterogeneity poses significant problems in the clinical use of chemosensitivity assays. The question arises as to whether a single biopsy specimen is representative of a patient's disease. In this last study, we sought evidence of tumor heterogeneity by comparing chemosensitivity responses between: 1) different portions of a single tumor, 2) a primary and a metastatic biopsy taken from a patient on the same day, and 3) different metastases from a patient taken on the same day. The results demonstrated the presence of considerable heterogeneity of response to chemotherapy among different tumors from the same patient, and even within the same tumor. The reported discrepancies of in vitro and in vivo sensitivity may be due to such therapeutic heterogeneity among tumors.