Use of the agar diffusion chamber for the exposure of human tumor cells to drugs.

Human melanoma xenografts in immune-deprived mice have been used to assess the value of the agar diffusion chamber for chemosensitivity testing. Tumor cells were treated with melphalan, Adriamycin, or methyl trans-1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea either as solid tumors growing in mice or as suspensions in agar in i.p. diffusion chambers. Survival of clonogenic human tumor cells was measured by the agar diffusion chamber assay in both cases. Cell survival curves were log-linear for treatment of tumor cells in vivo or in the chambers. For melphalan the slopes of survival curves were significantly greater for treatment in the chambers than as solid tumors in vivo, but for methyl trans-1-(2-chloroethyl(-3-(4-methylcyclohexyl)-1-nitrosourea or Adriamycin, they were indistinguishable. Experiments with [14C]melphalan showed that the levels of drug achieved were less inside the diffusion chambers than in the tumors in vivo so that the sensitivity of tumor cells to melphalan was much greater when they were treated in chambers. The differences in drug exposure and in cellular chemosensitivity between chambers and tumors suggest caution in the interpretation of drug testing using this system, but the log-linear nature of the dose-response curves is an important feature which may be useful in the eventual development of optimal chemosensitivity testing systems.

The therapeutic response of three human tumor lines maintained in immune-suppressed mice.

Studies were made of the growth and therapeutic response of three lines of human tumor serially transplanted in immune-suppressed mice. They included a well-differentiated colonic carcinoma (HX 13), a poorly differentiated colonic carcinoma (HX 18), and undifferentiated small-cell carcinoma of the bronchus (HX 29). Their histological appearance and growth rates were stable, with volume-doubling times ranging from 6 to 12 days. Studies by the technique of labeled mitoses showed that the growth kinetics of the three tumor lines were very similar, with median intermitotic times in the range of 26 to 35 hr. An analysis of the incidence of single and double takes revealed evidence for variation in susceptibility among the recipient mice. One tumor (HX 18) was transplantable with single-cell suspensions but 10(5) cells were required for 50 percent takes. The response of the tumors to a range of chemotherapeutic agents was studied. There was evidence that drugs that are known to be effective in the treatment of patients did well, in particular 5-fluorouracil against the colonic tumors and cyclophosphamide against the bronchial carcinoma. Long-term regressions induced by cyclophosphamide in the bronchial carcinoma may reflect assistance from host defense mechanisms.

Stem-cell survival and tumor control in the Lewis lung carcinoma.

The stem-cell response of the Lewis lung carcinoma to single doses of cyclophosphamide has been studied by three assay techniques: in vitro colony formation, lung colony formation, and the end-point dilution assay. These three techniques have given comparable results, and the end-point dilution results showed that 50 percent takes could be achieved with as few as 1 to 3 cells. Studies have been made of the growth of small i.m. implants and of the time following implantation at which they could be eradicated by cyclophosphamide. The results were compared with the curability that would be expected on the basis of cell survival studies. It was found that older (and therefore larger) implants were cured than might have been expected. It seems unlikely that this discrepancy was due to additional cell kill caused by an immune response. An alternative explanation, that the surviving fraction following a dose of cyclophosphamide was lower in small implants than in larger i.m. tumors, was supported by studies of cell survival in dissectable lung colonies.

Characterization of four new cell lines derived from human squamous carcinomas of the uterine cervix.

Four continuous cell lines were established from 15 biopsies of human squamous carcinomas of the uterine cervix, two from women less than 35 years old. All four lines grew as adherent monolayers and had epitheloid morphology. All required initial 3T3 feeder layer support and hydrocortisone and insulin for growth and have now been grown in vitro for at least 12 months. The individual lines possessed unique isozyme patterns and were distinct from the HeLa cell line. All were tumorigenic in nude mice. In vitro colony forming efficiencies ranged from 2 to 30% in a monolayer anchorage dependent assay but were only from 0.0025 to 0.6% when assayed in soft agar. The lines were all aneuploid with mean chromosome numbers ranging from 71 to 75. Analysis of intermediate filament expression showed that all lines were positive for cytokeratin expression and two were positive for vimentin expression. These low-passage cell lines represent a panel of new in vitro models of carcinoma of the cervix. They should be useful for the investigation of chemosensitivity, of the involvement of human Papillomavirus in this disease, and as models of squamous cell differentiation.

Tumor size dependency in the radiation response of the Lewis lung carcinoma.

Tumor cell survival characteristics were assessed following 60Co gamma-irradiation of the Lewis lung carcinoma as 500-cu mm s.c. tumors or as 0.5-cu mm (1 mm in diameter) pulmonary metastases. Cells in the small pulmonary tumors were markedly more radiosensitive (D0 = 106 rads; hypoxic fraction less than 0.005) than were those in large s.c. tumors (final D0, 315 rads; hypoxic fraction, 0.36). When pulmonary metastases were excised and irradiated intact under well-oxygenated conditions in vitro, the hypoxic fraction rose to 0.30. This implies that, under normal in situ conditions, these nodules contain a microvascular system that achieves adequate oxygen supply to the great majority of tumor cells. Thus, the tumor cells within these small metastatic implants were more sensitive to irradiation, largely due to better oxygenation, and may be more sensitive to chemotherapy, due to better drug availability.

DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cell line CHO-K1, has been demonstrated to be defective in DNA double-strand break repair and also in its proficiency to undergo V(D)J recombination. Recent work has provided both genetic and biochemical evidence that the M(r) 80,000 subunit of the Ku protein is able to complement the radiosensitivity and the V(D)J recombination defect in the xrs-6 mutant. We demonstrate here that complementation of the radiosensitive phenotype in xrs-6 cells by the introduction of Ku80 cDNA is accompanied by the concomitant restoration of DNA double-strand break rejoining proficiency to almost that of the parental CHO-K1 cells, as measured both by neutral single-cell microgel electrophoresis (Comet) technique and by pulsed-field gel electrophoresis. These results provide further biochemical evidence for the involvement of the Ku protein in the repair of DNA double-strand breaks.

Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67.

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.

Prospects for proton-beam radiotherapy.

Proton beams are already being employed for radiation therapy in 15 centres worldwide and over a dozen more are planned. Good clinical results have been reported in uveal melanomas and in sarcomas of the skull base. Calculated dose distributions for the treatment of brain, lung, head and neck and pelvic tumours predict an improvement relative to multiple-field or conformal photon radiotherapy. Protons may well provide high-precision radiotherapy that will lead to improved treatment of certain tumours in specific anatomical locations.

Differences in the level of DNA double-strand breaks in human tumour cell lines following low dose-rate irradiation.

It is now well accepted that differences exist in the intrinsic radiosensitivity of human tumour cells although the molecular basis of this is still unclear. Current evidence suggests that of the lesions induced in DNA by ionising radiation, double-strand breaks (DSB) are the most closely linked to cell death. In this study, levels of DSB were measured by neutral filter elution under conditions of both repair inhibition and maximum recovery and compared with clonogenic survival curves for high (HDR) and low dose-rate (LDR) irradiation in human carcinoma lines of differing radiosensitivity. Four human lung carcinoma lines were used, two small-cell (SCLC; HC12 and HX149) and two non-small cell lines (NSCLC; HX147A7 and HX148G7). Cell survival was measured by soft agar and monolayer colony-forming assays as appropriate and a large variation in sensitivity of the cell lines was seen (alpha values of 0.06 to 0.56 Gy-1). We have previously reported that the damage induced at high dose rate does vary in these cell lines but not in a way which correlates with their cell survival response [5]. Following irradiation to 15 Gy at low dose rate essentially no DSBs were detected in any of the four lines but at 70 Gy the more sensitive SCLC showed more residual damage than in the more radioresistant NSCLC lines. The prime determinant of the difference between the LDR and HDR damage curves is likely to be repair occurring during irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung damage in mice from cyclophosphamide and thoracic irradiation: the effect of timing.

The development of lung damage in mice following thoracic irradiation is enhanced by cyclophosphamide (CY). CY was given at various times from 28 days before to 28 days after irradiation. Although increased damage was seen at all times, the extent showed marked variation according to the time interval. The most marked variations were seen when CY was given within 24 hours of irradiation; maximal enhancement was seen when CY was given before or with irradiation and minimal response when given 12 hrs before or 12 hrs after irradiation. It may therefore be prudent for the clinician to avoid such close time intervals between cytotoxic drugs and irradiation, unless a specific time-related therapeutic gain can be exploited.

Differential radiosensitization by the poly(ADP-ribose) transferase inhibitor 3-aminobenzamide in human tumor cells of varying radiosensitivity.

Four newly-established human tumor cell lines, have been irradiated at dose rates of 150 and 3.2 cGy/min to compare their capacity to repair radiation damage. They included a neuroblastoma, a germ-cell carcinoma of the testis, a large cell carcinoma of the lung, and a carcinoma of the cervix. The four lines varied in their sensitivity to high dose-rate irradiation, with the neuroblastoma being most radiosensitive and the lung and cervix tumors the most radioresistant. The extent of dose sparing associated with lowering the dose rate to 3.2 cGy/min was similar in three of the lines but somewhat greater in the case of the cervix carcinoma cell line. The presence of non-toxic concentrations of the poly(ADP-ribose) transferase inhibitor, 3-aminobenzamide (3-AB), enhanced the response of 3 of the 4 tumors to irradiation; it failed to modify the sensitivity of a lung carcinoma cell line. The extent of sensitization was generally similar at high and low dose rate. Measurement of poly(ADP-ribose) transferase activity in control and irradiated cells showed the neuroblastoma cells to contain much higher initial levels than the other three lines but there were no significant differences in the extent of stimulation in enzyme levels after irradiation. Survival curves obtained at low dose-rate help define the initial slope of the acute curve and it appears that 3-AB may exert a differential effect among human tumors in modifying this component.

Survival of murine clonogenic stem spermatogonia following combined radiation and cytotoxic drug treatments.

The effects of combined radiation and cytotoxic drug treatments on the survival of murine clonogenic stem spermatogonia have been investigated using an in vivo clonogenic assay. Drug doses (BCNU, 15 mg/kg; cyclophosphamide (CY) 150 mg/kg, and procarbazine (PCB) 200 mg/kg) were chosen to kill a high proportion of differentiated spermatogonia, but no stem-cells, and were administered as single doses up to 14 days prior to or after 9 Gy of X rays. BCNU produced a moderate enhancement of damage (DEF, 1.0-1.3) that was relatively time-independent. Both CY and PCB demonstrated considerable enhancement of damage (DEF, 1.6 +/- 0.03, respectively) when administered 1 day prior to radiation, and also 3 days prior to radiation for PCB (DEF = 1.4 +/- 0.05). The enhancement of damage may have been caused by either a change in the shoulder width of the cell-survival curve, or additive cell-killing as indicated by a dose-response study.

Treatment results and prognostic factors in 101 men treated for squamous carcinoma of the penis.

PURPOSE: This retrospective study was performed to assess the treatment outcome and prognostic factors in 101 men with invasive squamous carcinoma of the penis treated at the Royal Marsden Hospital between 1960-1990. METHODS AND MATERIALS: The tumor was confined to the glans penis (T1) in 79 patients, 82 were node negative (N0), and two patients had distant metastases at presentation. The histology was Grade 1 (G1) in 36, Grade 2 (G2) in 18, Grade 3 (G3) in 28, and unknown in 19 patients. Node-positive disease was commoner in patients with G3 (p = 0.02) or T2/3/4 tumors (p = 0.007). Treatment for the primary tumor was external beam radiotherapy (EBRT) in 59, interstitial brachytherapy in 13, and partial or total penectomy in 29 patients. The median dose, dose/fraction, and treatment time for EBRT was 60 Gy, 2 Gy/fraction, and 46 days, respectively. Eighty patients received no inguinal node treatment, 13 had EBRT (4 with chemotherapy), and 8 underwent groin dissection at presentation. RESULTS: During a median follow-up of 5.2 years (2 months-22 years), 56 patients died (penile cancer 31, intercurrent illness 23 and unknown cause 2), giving 10 year overall and cause-specific survival (CSS) of 39 and 57%, respectively. Adverse prognostic factors for CSS on univariate analysis were G3, ulcerative/fungating or T2/3/ 4 tumors, node positive, Jackson's Stage 2/3/4, and surgical treatment for the primary. All but the last two were significant independent prognostic factors for CSS on multivariate analysis. Penile or perineal recurrence or residual disease after initial treatment was seen in 36 out of 98 evaluable patients, giving a 10-year local failure rate (LFR) of 45%. Local failure after initial treatment was successfully salvaged in the majority (26 out of 36) of patients with further surgery or radiotherapy, and local control was achieved ultimately in 74 out of 77 T1, 7 out of 12 T2; 3 out of 3 T3, and 3 out of 5 T4 tumors. In the 44 evaluable patients with T1 tumors treated by EBRT the only adverse RT parameter approaching prognostic significance (p = 0.052) was a BED value corrected for recovery of <60 Gy (alpha/beta 10, K = 0.5 Gy/day, mean = 21 days). CONCLUSION: Invasive squamous carcinomas of the penis carry a significant risk of loco-regional recurrence after initial radiotherapy and this can be successfully salvaged in most patients with further treatment. This mandates close follow-up to detect loco regional recurrence early.

The effect of low dose-rate and cyclophosphamide on the radiation tolerance of the mouse lung.

Mice were given cyclophosphamide 30 mg/kg intraperitoneally before thoracic irradiation at high dose-rate (HDR, 100 cGy/min) or low dose-rate (LDR, 5 cGy/min). The development of pneumonitis was monitored by regular measurement of breathing rate. Peak rises in breathing rate occurred around 4-9 weeks in those given cyclophosphamide before irradiation, and around 14-16 weeks in those given radiation alone, regardless of dose-rate. The dose reduction factor (DRF = LDR/HDR ratio) for LDR irradiation was congruent to 1.8 but LDR sparing was abolished (DRF congruent to 1.0) when cyclophosphamide was given before irradiation.

Continuous irradiation of the Lewis lung carcinoma in vivo at clinically-used "ultra" low-dose-rates.

The in vivo response of the transplantable Lewis lung tumor to continuous irradiation (CI) at the clinical "ultra" low-dose-rates of 10-30 cGy/h was investigated. Tumor-bearing mice were housed in a dedicated 137Cs unit for up to three weeks while receiving CI. The horizontal 137Cs beam allowed secondary shielding of the lower body, but full irradiation of the dorsally-placed tumor and part of the upper body. Radiation survival curves were measured using an excision assay and tumor-cell colony formation in soft agar. The relative cell survival per tumor decreased exponentially with time and the irradiation periods required to reach a surviving fraction of 10(3) at the three dose-rates (10, 15 and 30 cGy/h) were 15.4, 10.2 and 4.3 days. As a function of dose, the survival curves were exponential and indistinguishable between the three dose-rates, the average DO value being 5.04 Gy. Neither the hypoxic fraction of the tumor nor the intrinsic cellular radiosensitivity were altered in the tumor cells surviving CI. The effects of the three dose-rates on the growth of Lewis lung tumors differed considerably, with clear volume regression at the highest dose rate (30 cGy/h) and exponential decreases in clonogenic cells per tumor with time at 15 and 30 cGy/h. Since the overall effect of CI is determined by the competition between cell killing and cell repopulation, the response of slowly-growing human tumors should be proportionately greater.

Adriamycin sensitivity following "ultra" low dose rate irradiation of Lewis lung carcinoma in vivo.

As an extension of recent study on the response of the Lewis lung tumor to low dose rate continuous irradiation (CI) at 15 cGy/hr, we have gone on to investigate the effects of such irradiation on the sensitivity of tumor cells to treatment with Adriamycin (Adr). Cells from untreated tumors gave an exponential dose response curve to Adr in vitro, the D10 of which increased (sensitivity decreased) with the size of tumor (0.05 g to 0.6 g) from which the cells were obtained. After previous in vivo CI to a total dose of 28 Gy (irradiation time--186 hr), this size dependence was abolished and the cells showed an exponential response to Adr in vitro (D10 = 0.4 microgram/ml). The enhancement was also observed after equivalent doses of fractionated irradiation, but not after acute irradiation. It was difficult to characterize the proliferative status of the cells surviving irradiation, but repopulation studies showed that only after CI was there any delay before repopulation commenced. LL was relatively insensitive to Adr in vivo, however, we did observe an increased effect after previous CI.

Heterogeneity of radiosensitivity in a human glioma cell line.

Sixteen clones were isolated from an early-passage human glioma cell line (IN859) and have been found to show variation in several biological characteristics including DNA content, modal chromosome number, and morphology. In addition, heterogeneity of radiosensitivity was detected: the doses that gave a surviving fraction of 0.01 varied by a factor of approximately 1.5. The most sensitive (clone 6) and the most resistant (clone 9) clones were selected for further study; their surviving fractions at 2Gy (SF2) were 0.37 and 0.64, respectively. When compared at a fixed radiation dose the sensitive clone surprisingly demonstrated greater split-dose recovery than the resistant clone; it also showed greater low dose-rate sparing.

The effects of anesthetics and misonidazole on the development of radiation-induced lung damage in mice.

The measurement of breathing frequency as a functional end-point of radiation-induced lung injury in mice allowed two phases of damage to be discerned; the first was manifest at 12-20 weeks after irradiation, the second beyond 28 weeks. Anesthesia by pentobarbitone sodium or steroids gave significant radioprotection of the lung during the early pneumonitic phase. Addition of the hypoxic cell sensitizer misonidazole removed the protective influence of the anesthetics but did not sensitize the lungs of unanesthetized mice. No anesthetic protection was detected for the late response, showing evidence for dissociation between early and late lung damage. The degree of epilation was measured on the dorsal thoracic region of the same mice. Protection by anesthetics and its reversal by misonidazole was also demonstrated. These results provide a warning of potential hazards in the laboratory evaluation of chemical radiosensitizers. The use of anesthetics at the time of irradiation could lead to an exaggerated enhancement of normal tissue damage.

The search for therapeutic gain in the combination of radiotherapy and chemotherapy.

The literature on combined treatment with radiation and cytotoxic drugs in experimental tumours and normal tissues of laboratory animals is reviewed in the context of the four previously proposed mechanisms whereby a therapeutic advantage might be gained. There is evidence for strong time-dependent processes occurring in some normal tissues. In tumours, the evidence for this is much weaker and there is considerable disparity among experimental tumours in optimum timing. This review leads to the conclusion that the clinical use of drug-radiation combinations should not be based on an anticipated beneficial interaction; gain will most probably come from the best radiotherapy and the best chemotherapy given as far as possible independently. Deleterious interactions can be reduced by allowing a gap of some weeks between chemotherapy and radiotherapy and by avoiding drugs that are known to enhance radiation damage to the normal tissues that are irradiated.

The ESTRO Breur lecture. Cellular sensitivity to low dose-rate irradiation focuses the problem of tumour radioresistance.

This paper emphasises the radiobiology of human tumour cells irradiated at the relatively low dose rate of 1 Gy/h (i.e.1-2 cGy/min), described here as the "Regaud dose rate". Continuous irradiation at this dose rate is approximately isoeffective with fractionated radiotherapy using 2 Gy/fractions. At the Regaud dose rate, cell survival curves are approximately exponential and they appear to extrapolate the initial slope of the high dose rate survival curve. Little recovery occurs after such treatments since it has largely taken place during irradiation. At the Regaud dose rate human tumour cell lines show a wide range of radiosensitivities, differing by a factor of around 7. This may well be the most clinically-relevant way of describing the radiosensitivity of tumour cells. Current models of radiation cell killing envisage a component of damage that increases linearly with dose. It is this component that dominates the slope of the Regaud survival curve. It may be produced by DNA damage due to clusters of ionisation events, or perhaps by damage to hypersensitive parts of the genome. The steepness of this component of damage may be modified by exogenous inhibitors of DNA damage repair.