Glucose starvation and acidosis: effect on experimental metastatic potential, DNA content and MTX resistance of murine tumour cells.

Exposure to oxygen deprivation in vitro has been reported to cause drug resistance in CHO cells (Rice et al., 1986; PNAS 83, 5978) and enhancement of experimental metastatic (colonisation) ability of murine tumour cells (Young et al., 1988; PNAS 85, 9533). Both these studies also demonstrated the induction of a subpopulation of cells with excess DNA content. Since the micromilieu in tumours results in exposure of the tumour cells to conditions of acid pH and nutrient deprivation, as well as hypoxia, we have examined the effect of exposure to acidosis (pH 6.5) and glucose starvation on drug resistance, cellular DNA content and the experimental metastatic ability of KHT sarcoma and B16F1 melanoma cells. Cells were exposed to these conditions for 24 and 48 h and tested for resistance to methotrexate (MTX) or experimental metastatic ability either immediately following these exposures or after 24 or 48 h of recovery in normal growth medium. Both cell lines demonstrated an enhancement of colonisation potential, which was most marked when cells were injected after 48 h of exposure followed by a 24 or 48 h recovery period. Flow cytometric analysis demonstrated an increase in the fraction of KHT cells with excess DNA following both glucose starvation and acidosis we observed only a small increase in MTX resistance following acidic exposure of cells and no change following glucose starvation. Since both acidosis and glucose starvation are known to induce glucose regulated proteins (grp), a subset of the stress protein family, we studied the effect of treatment with another known inducer, 2-deoxyglucose. We found that this agent affected the metastatic efficiency of KHT cells in a manner similar to that observed following exposure to glucose starvation and acidosis. However, further studies are required to establish what role, if any, grp play in this effect. In conclusion this study shows that transient exposure of murine tumour cells to an acidic or glucose deprived environment can cause progression in terms of metastatic potential.

Lack of radiosensitization in Lewis lung carcinoma and a murine plasmacytoma by MTDQ and MTDQ-DA.

MTDQ, and its watersoluble derivative MTDQ-DA, have been tested in combination with radiation using the murine plasmacytoma X5563 and the Lewis lung carcinoma. Neither of the two compounds showed any effect on the single dose radiation response as measured by tumour growth delay. In addition no effect of MTDQ-DA on the response of the X5563 plasmacytoma to fractionated radiation was observed. As a control, studies with the established hypoxic cell sensitizer Misonidazole showed that both tumours could be sensitized to radiation by that drug, in the case of Lewis lung carcinoma the use of the excision cell survival assay showed that this was due to hypoxic cell sensitization.

Response of the seminiferous epithelium to scattered radiation in seminoma patients.

Semen and blood samples were obtained, at 3-month intervals over 12 to 28 months, from patients who underwent subdiaphragmal radiation after orchidectomy for seminoma testis. Before radiotherapy a mean (+/- SE) semen volume of 4.7 +/- 0.5 ml, a mean sperm count of 44.4 +/- 13.5 x 10(6)/ml, a mean percentage of motile cells of 20.3 +/- 5.2, a mean percentage of morphologically normal spermatozoa of 13.4 +/- 5.4, a mean percentage of swollen sperm of 39.6 +/- 7.4, and a mean serum follicle-stimulating hormone (FSH) value of 8.3 +/- 1.2 mIU/ml was found. The mean testicular dose from scatter was 62 +/- 5 cGy (range, 34 to 95 cGy). Sperm counts between 0 and 2.75 x 10(6)/ml were seen at 6.8 +/- 0.6 months and recovery to values greater than 2.25 x 10(6)/ml at 11.8 +/- 0.8 months after the start of radiation. Peak FSH values of 19.2 +/- 1.6 mIU/ml were obtained at 6.7 +/- 0.9 months after the start of irradiation. After recovery mean semen volume was 3.9 +/- 0.4 ml, mean sperm count 34.6 +/- 5.6 x 10(6)/ml, the mean percentage of motile cells 42.5 +/- 6.0, the mean percentage of swollen sperm 58.7 +/- 6.8, and the mean percentage of spermatozoa with normal morphology 23.4 +/- 5.1. Only motility was significantly different (P less than 0.01) from pretreatment values. The elevation of FSH values with time after start of radiotherapy reflected the toxicity to spermatogenesis but no correlation was found between peak FSH levels and scattered radiation dose. Also, neither the time from start of radiotherapy to sperm count nadir or recovery nor the time to peak FSH levels was significantly correlated with radiation dose.

The clinical course of lung metastases from breast cancer.

The clinical course of 50 breast cancer patients whose first metastases were found in the lungs was investigated. 18 months after the start of primary treatment 50% of the patients had developed pulmonary metastases (range: 0-81 months). In 23% of patients a solitary, and in 68% more than one, lung metastases were detected. After a median time of 4 months, in 56% of patients the disease had spread to further organs with bone (25%) and liver (17%) being the most frequent sites. First line management of lung metastases employed surgery, endocrine treatment, chemotherapy or a combination of these modalities. In 24% of patients a complete response was achieved, and in 11% a partial response, with an overall response rate of 35%. Median survival from detection of lung metastases was 13 months (range 4-123+). Patients with only a solitary lung metastasis survived for a median of 11.5 months as compared to 10.5 months for patients with more than one pulmonary metastases. Patients with a disease-free interval of more than 18 months survived significantly longer than patients with a disease-free survival of 18 months or less.

Protection from radiation-induced damage to spermatogenesis in the androgen pretreated rat.

Protection of spermatogenesis from radiation-induced damage has been investigated in the adult Wistar rat. Silastic tubing containing either cholesterol or testosterone was implanted subcutaneously 7 weeks before 4 equal daily fractions of either 1, 1.5, 2, and 2.5 Gy of 230 kVp X-rays locally to the testes. Implants were removed on the day following the last fraction and 8 weeks after irradiation 88.6%, 83.8%, 63.6% and 28.9% tubule cross-sections respectively were found regenerating in rats pretreated with testosterone. In contrast, 68.45%, 58.6%, 38.2% and 17.3% tubule cross-sections regenerating were obtained in rats pretreated with cholesterol. Changes in testis weight however were found to show the reverse trend (i.e. a greater weight loss was observed following androgen pretreatment). These results show that protection of spermatogenesis from fractionated irradiation may be achieved in rat testis by androgen pretreatment.

Growth and chemotherapy of a human germ-cell tumour line (GCT 27).

The human germ-cell tumour cell line GCT 27 growing as subcutaneous xenograft tumours in male nude mice was used in the 4th and 5th passage to study chemotherapeutic drug responses. Recipient mice received 5 Gy whole body irradiation immediately before tumour transplantation. The median take rate was 62% (range 39-73%) and the median volume doubling time 14 days (range 7-28 days). For bleomycin, cisplatin and carboplatin a clear dose response for growth delay was observed. Bleomycin caused substantial weight loss at doses above 75 mg/kg whereas good response to cisplatin was obtained without serious toxic effects. Vinblastine and etoposide exerted no effect when given in non-toxic doses. The response to etoposide was not improved either by fractionated treatment or by combination with verapamil. However, the combination of 20 mg/kg etoposide and 2 mg/kg cisplatin, which when given alone were ineffective, led to a growth delay that was equal to that observed following the administration of higher cisplatin doses. This effect may be explained by the fact that etoposide, as an inhibitor of DNA-topoisomerase II, may interfere with the repair of DNA interstrand cross-links caused by cisplatin.

Comparative effects of X irradiation on the testes of adult Sprague-Dawley and Wistar rats.

The response of the testes of two strains of adult rats (Sprague-Dawley and Wistar) to graded single doses and split doses of 230 kVp X rays has been investigated. A marked difference was noted between the strains in the response of the clonogenic spermatogonia to irradiation, as measured histologically by the repopulation index. Single-dose response curves derived for these cells in the Sprague-Dawley strain had a much larger shoulder (up to about 4-5 Gy) than for the Wistar (less than 2 Gy). Split-dose studies revealed that this difference may partly be explained by a greater repair capacity in the cells of the Sprague-Dawley strain. Changes in serum FSH concentrations mirrored the changes in clonogenic spermatogonial survival following split doses of radiation.

The response of murine stem spermatogonia to radiation combined with 3-aminobenzamide.

The influence of 3-aminobenzamide (3-AB) on the radiation response of the stem spermatogonia of the CBA mouse has been investigated. Doses of 3-AB from 66 to 450 mg/kg, administered 1 h before irradiation, significantly enhanced stem-cell killing. Enhancement was observed when 3-AB (450 mg/kg) was given up to 5 h before, but not if administered after, irradiation. When radiation was delivered at a lower dose rate (5 cGy/min compared to 180 cGy/min) significant dose sparing was achieved for radiation alone. Pretreatment with 3-AB resulted in slightly less enhancement at the low dose rate than at the high. Split-dose studies (9 Gy total dose) with radiation alone resulted in a recovery ratio of 1.4-1.5. Administration of 3-AB before the first dose resulted in a similar recovery ratio, but if given immediately after the first dose the ratio was smaller. Pretreatment of mice with the radiosensitizer RSU-1069 indicated that at least some of the stem cells were radiobiologically hypoxic. We suggest therefore that the enhancement of spermatogonial stem-cell killing by 3-AB is not entirely due to inhibition of repair processes but may also involve modification of the oxygen status of the testis.

Effect of dose-rate on the survival of murine spermatogonia following 60Co gamma-irradiation.

The survival of murine clonogenic stem and differentiated spermatogonia was assessed by counts of repopulating tubules in histological sections at 35 days and counts of testicular sperm-heads at 29 days respectively, following irradiation delivered at five dose-rates (1.1, 2.5, 5, 11 and 112 Cgy X min-1). For stem spermatogonia, a significant increase in cell survival accompanied the reduction in dose-rate, and this was characterized by an increase of the Do for the derived survival curves from 2.86 +/- 0.29 Gy at 112 cGy X min-1 to 4.46 +/- 0.45 Gy at 1.1 cGy X min-1. The data were analysed using two mathematical models for repair at varying dose-rates, the LPL model of Curtis and the incomplete repair model of Thames. Using these models the half-time for repair was estimated to lie between 15 and 25 min. No dose-rate effects were observed for the differentiated spermatogonia. The sparing of stem spermatogonia at reduced dose-rate, may have implications for the future fertility of men treated with low dose-rate total body irradiation (TBI).

Attempted protection of spermatogenesis from single doses of gamma-irradiation in the androgen pretreated rat.

Spermatogenic stem-cell survival after gamma-irradiation has been investigated in the adult Wistar rat. Single doses of 4.5 and 9 Gy gamma-rays were administered to the testes of rats who received arachis oil (0.1 ml/100 g body weight) or testosterone enanthate (240 micrograms/100 g body weight) subcutaneously three times weekly for 6 weeks prior to radiation and during the week in which the radiations were given. A mean percentage of regenerating seminiferous tubule cross-sections of 32.45% and 7.26% was found in the testes of androgen-pretreated rats at 8 weeks after 4.5 and 9 Gy, respectively. Similar values (33.4% and 6.2%) were obtained in arachis oil-pretreated controls. We therefore conclude that protection of rat spermatogenesis from single doses of gamma-rays cannot be achieved by androgen pretreatment.

[Radiotherapy of carcinoma of the paranasal sinuses]. / Strahlentherapie der Nasennebenhöhlenkarzinome.

Carcinomas of the paranasal sinuses are usually advanced when diagnosed and present a therapeutic challenge. During the period between February 1970 and June 1981 44 patients were treated. 22 received postoperative irradiation, seven in combination with chemotherapy. 18 patients were treated with radiation alone, eleven with concomitant chemotherapy. Four patients received preoperative irradiation, three in combination with chemotherapy. The three-year survival is 43% and the five-year survival 33%. For those 26 patients who were irradiated pre- or postoperatively with or without concomitant chemotherapy the five-year survival is 45%. We believe the patient will be afforded the greatest opportunity for cure with the combined efforts of the radiotherapist and the surgeon. The combination of chemotherapy and radiotherapy did not provide better results but increased acute and chronic toxicity of the therapy.

Report of two cases of male breast cancer after prolonged estrogen treatment for prostatic carcinoma.

Reviewing our experience with 19 cases of male breast cancer, we found two patients with a 12-year history of estrogen therapy for prostatic carcinoma. One initially received diethylstilbene followed by estradiol, 80 mg monthly, and finally estramustin, 140 mg twice daily. The other patient had dienestrol, 2.5 mg daily, and later estradiol, 40 mg every 6 weeks. The possible causal relationship between prolonged estrogen administration and breast cancer in males is in contrast to available epidemiologic data. Therefore, the possibility of an association by chance cannot be ruled out.

[Male breast cancer]. / Das männliche Mammakarzinom.

Between 1974 and 1982 inclusive 18 male patients were treated for breast carcinoma. 12 patients had postoperative radiotherapy whereas 4 were referred for treatment of recurrent or metastatic disease. One patient showed signs of inflammatory breast cancer and was treated with chemo-radiotherapy and one was being followed up in our department after radiotherapy for prostatic cancer in 1970. Median overall survival was 52 months and the median disease-free interval was 21 months.