Improved treatment of a brain-tumor model. Part 1: Advantages of single- over multiple-dose BCNU schedules

Clonogenic cell and animal survival studies were used to determine the most effective BCNU therapy schedule in the 9L rat brain-tumor model. Survival of tumor cells following a single LD10 dose of BCNU (13.3 mg/kg intraperitoneally) was compared to cell survival after one to four daily 0.5 X LD10 doses. The posttreatment kinetics of surviving clonogenic cells were investigated at various times after BCNU was given in single doses of 0.25 to 1 X LD10 and in two daily doses of 0.5 X LD10. The cell kill was greater, time to reinitiation of cell growth was later, posttreatment rate of clonogenic cell proliferation was slower, and the interval to total repopulation of the clonogenic cell pool was longer with a single LD10 dose as compared to the multiple-dose schedules. Animal survival studies confirmed that a single LD10 dose of BCNU was at least as effective as a cumulative level of up to 1 1/2 times that amount when treatment was administered in smaller doses, regardless of the fractionation schedule. Clinical experience with patients harboring malignant brain tumors has shown that a single BCNU dose of 185 to 200 mg/sq m is tolerated well. Results of these animal experiments suggest that this therapy should have anti-tumor activity at least equivalent to the more commonly employed schedule of 80 mg/sq m/day given for 3 days. Although direct comparison of treatment efficacy using the two schedules is not possible, no adverse clinical effects have been observed with the recently adopted single-dose schedule. Furthermore, the duration of patient hospitalization for chemotherapy has decreased

Investigation of resistance to DNA cross-linking agents in 9L cell lines with different sensitivities to chloroethylnitrosoureas

The 9L-2, 9L-7, and 9L-8 cell lines, derived from the 9L in vivo rat brain tumor, were treated with nitrosoureas that can alkylate and cross-link DNA and carbamoylate intracellular molecules to various extents. Compared to 9L cells, 9L-2 cells were very resistant to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, and to 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-deoxyglucopyranose. The sensitivity of 9L-7 and 9L-8 cell lines to these drugs was intermediate between 9L and 9L-2. Treatment of 9L, 9L-2, 9L-7, and 9L-8 cell lines with 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea produced approximately the same level of cell kill. Compared to 9L cells, 9L-2 cells are 10-fold more resistant to the cytotoxic effects, 34-fold more resistant to the induction of sister chromatid exchanges, and have 40\% fewer DNA interstrand cross-links caused by treatment with 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea . In contrast, treatment of 9L and 9L-2 cells with 1-ethylnitrosourea produced approximately the same level of cell kill and induction of sister chromatid exchanges. Our results suggest that the resistance of 9L-2, 9L-7, and 9L-8 cells is related to DNA cross-linking and not to alkylation or carbamoylation. We studied the effects of other agents that form DNA cross-links with structures different from those formed by treatment with chloroethylnitrosoureas (CENUs) in 9L and 9L-2 cells. In contrast to results obtained with CENUs, 9L-2 cells were 2-fold more sensitive to the cytotoxic effects, 2-fold more sensitive to the induction of sister chromatid exchanges, and had 3-fold more cross-links formed than 9L cells treated with nitrogen mustard. However, the amount of cell kill, number of sister chromatid exchanges induced, and the DNA cross-linking were the same for 9L and 9L-2 cells treated with cis-diamminedichlorplatinum(II). Our results indicate that cellular resistance to CENUs is highly specific and that the mechanism of resistance does not allow cross-resistance with other DNA cross-linking agents. These and other results suggest that when DNA repair processes mediate cellular resistance to CENUs, other cross-linking agents will not be cross-resistant unless they form alkylation products that are affected by repair processes that mediate resistance to CENUs

Prediction of the relative in vitro sensitivity of 9L rat brain tumor cells to nitrosoureas by the sister chromatid exchange assay

In an earlier study we showed that there is a good correlation between sister chromatid exchange induction and cell kill in 9L cells treated with certain nitrosoureas. In the study reported here, we treated four 9L cell lines that have different sensitivities to chloroethylnitrosoureas with 1,3-bis (2-chloroethyl)-1-nitrosourea, chlorozotocin, and ethylnitrosourea and determined the number of sister chromatid exchanges induced. Cell lines that were most sensitive to the drugs with respect to cell kill were also most sensitive to induction of sister chromatid exchanges for a given drug, and the assay based on sister chromatid exchange is therefore predictive of the relative sensitivity of these cells to the drugs used

Stem cell studies of human malignant brain tumors. Part 1: Development of the stem cell assay and its potential

A stem cell assay for human malignant gliomas has been developed. Cells obtained from tumor biopsies grew into colonies composed of malignant glial cells, as documented by histochemical, immunohistochemical, and immunobiological techniques. Studies suggest that the disaggregated cells are representative of the cells within the solid tumor. Clonogenic cells were obtained from 48 tumors and analyzed for their in vitro sensitivity to graded doses of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The in vitro anti-tumor activity of BCNU at clinically achievable doses was compared to clinical response to the agent based on changes in computerized tomographic scan, radionuclide brain scan, and neurological examinations. Twenty-two patients received nitrosoureas before or after tumor specimen analysis, and were eligible for in vitro-in situ correlations. Clinical tumor sensitivity to nitrosoureas was predicted by culture results in 42\% of all evaluable patients, and clinical resistance was predicted in 100\%. The capability of the assay can be appreciated best for the 13 patients not treated with BCNU prior to culture; the in vitro prediction of clinical sensitivity and resistance was 71\% and 100\%, respectively. Preliminary findings show that clinical tumor resistance to BCNU may result from "intrinsic" cell resistance in some patients and from inadequate delivery of drug to tumor cells in other cases. The potential utility of this method to study the reason(s) for tumor cell resistance to drugs, to screen new chemotherapeutic agents, to individualize patient treatment, and to investigate tumor biology is discussed

Age-related chemosensitivity of stem cells from human malignant brain tumours

After radiation therapy and chemotherapy for malignant glioma, patients aged 50 or under survive longer than patients over 50. Data from Brain Tumor Study Group trials show that, without treatment, these age groups have similar survival; therefore unperturbed tumour growth does not account for the difference. Sixteen consecutive patients with malignant glioma were studied, half of whom were less than or equal to 50 years of age; none had been treated before initial surgery, and all were subsequently treated with radiation and chemotherapeutic agents (in all but two patients, with nitrosoureas). Median survival of those aged greater than 50 was less than or equal to 50 years was 54 + weeks whereas that of those aged greater than 50 was 37 weeks. The longer survival for younger patients could not be attributed to tumour type, size, or location, pretreatment Karnofsky status, or mode of treatment. In-vitro sensitivity testing of clonogenic cells obtained from biopsy specimens showed that tumour cells from seven of eight patients aged less than or equal to 50 years were sensitive to 1,3-bis (2-chloroethyl)-1-nitrosourea (greater than 40\% cell kill at clinically achievable concentrations) compared with only one patient with sensitive cells out of eight older patients. Patient age was inversely correlated with in-vitro cell kill, and patients with sensitive cells were significantly younger than those with resistant cells. Therefore influence of age on survival after treatment of malignant gliomas is probably due to inherent differences in the sensitivity of clonogenic cells to radiation and/or chemotherapeutic agents