Analysis of ascites from patients with ovarian carcinoma by cell flow cytometry.

Cell flow cytometry offers the opportunity to analyze cytopathological samples with regards to DNA content and proliferative activity. To investigate whether this modality can quantitate certain aspects of ovarian carcinoma by analyzing ascites, 43 samples from patients with advanced papillary serous adenocarcinoma of the ovary were studied. In 28 samples (65%) ploidy and the percentage of cells in S phase (%S phase) could be analyzed. Fifteen samples could not be analyzed because of overlapping cell populations distorting distinct cell cycle phases. Of the 28 samples studied, 8 (29%) were diploid and 20 (71%) were aneuploid. The DNA in aneuploid samples ranged from 1.23 to 2.65. The %S phase for aneuploid was greater than that for diploid samples. Patients with diploid samples survived longer. Cytometric analysis of cells from ascites in 4 patients in whom disease progressed after they received chemotherapy showed that the percentage of cells in S phase increased. Cells from ascites established in vitro showed that ploidy and proliferative activity changed as cells were passed in culture. In conclusion, the analysis of ascites by cell flow cytometry may be a prognosticator in patients with advanced ovarian carcinoma. In addition, conclusions extrapolated from in vitro data to the in vivo situation should be done cautiously since late-passaged cells may not always be representative of the initial tumor sample.     

Biased immunoglobulin variable region gene expression by Ly-1 B cells due to clonal selection

Most, if not all, autoantibodies specific for bromelain-treated mouse erythrocytes recognize the common membrane phospholipid, phosphatidyl choline (PtC). Anti-PtC antibodies are produced by 5%-15% of CD5+ Ly-1 B cells of normal unimmunized mice, but not by detectable numbers of conventional CD5- B cells. At 1 week of age PtC-specific B cells are undetectable but then increase dramatically over the next 3 to 4 weeks to reach adult numbers. We report here that PtC-specific Ly-1 B cells in B10.H-2aH-4bp/Wts mice predominantly express either of two heavy and kappa chain variable (V) region gene combinations. In addition, the sequence and length of DH genes are conserved among cells expressing the same V gene combination, and the V kappa-J kappa junctions of one group involve unusual splice sites. Preferential V gene rearrangement models are insufficient to explain the DH and V kappa-J kappa junctional sequences or the delayed appearance of this specificity, and so they cannot solely account for the high frequency of PtC-specific cells. These characteristics are more consistent with antigen selection. We therefore attribute the frequent use of the two V region gene combinations to selection for cells that express them and conclude that the expressed V gene repertoire of Ly-1 B cells in adult mice is influenced by antigen selection. Apparently, there is no selection for mutant anti-PtC antibodies of higher affinity during the formation of the Ly-1 B repertoire because the V region genes expressed by PtC-specific cells are unmutated. Our findings are consistent with an important, germ line-encoded function for the immunoglobulin products of these gene combinations.     

The radioprotector WR1065 reduces radiation-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells

N-(2-mercaptoethyl)-lt3-diaminopropane (WR1065) protects againstradiation-induced cell killing and mutagenesis at the hypoxanthine-guaninephosphoribosyl transferase (HGPRT) locus in V79 Chinese hamsterhing fibroblast cells. At a concentration of 4 mM, WR1065 wasfound to be effective in protecting against radiation-inducedcell lethality only if present during irradiation, e.g., a dosemodification factor (DMF) of 1.9. No protective effect was observedif the protector was added within 5 min after irradiation or3 h later, e.g., DMFs of 1.0 and 1.1, respectively. The effectof WR1065 on radiation-induced mutation, expressed as resistanceto the cytotoxic purine analogue 6-thioguanine (HGPRT), wasalso investigated. In contrast to the treatment-schedule dependencefor protection by WR1065 against cell killing, this agent waseffective in reducing radiation-induced mutations regardlessof when it was administered. Following a dose of 10 Gy of ⁶⁰Co-rays, the mutation frequencies observed per 10⁶ survivors were77 ± 8, 27 ± 6, 42 ± 7, and 42 ±7 for radiation only, and WR1065 present during, immediatelyafter, or 3 h after irradiation. These data suggest that althougha segment of radiation-induced damage leading to reproductivedeath cannot be modulated through the postirradiation actionof WR1065, processes leading to the fixation of gross geneticdamage and mutation induction in surviving cells can be effectivelyaltered and interfered with leading to a marked reduction inmutation frequency.     

The effects of cycloheximide and WR-1065 on radiation-induced repair processes: a mechanism for chemoprevention

The effects of cycloheximide (CHX) and 2-[(aminopropyl)-amino]ethanethiol(WR-1065), each alone or in combination, on radiation-inducedmutation induction at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus and cell killing were investigatedusing a Chinese hamster ovary (CHO) AA8 cell system. Treatmentwith CHX, a potent inhibitor of protein synthesis, at a concentrationof 10 µg/ml administered 30 min prior to irradiation with7.5 Gy had no effect on cell survival but did reduce the radiation-inducedmutation frequency (per 10⁶ survivors) from 106.5±8.8(SEM) to 36.2±5.6 (SEM). Exposure of cells to 4 mM WR-1065reduced the mutation frequency to 44.8±4.2 (SEM), butthe combination of agents afforded no additional protection,that is 41.1±3.3 (SEM). The mechanism of action attributedto CHX in reducing mutation frequency is its ability to preventthe induction of an error-prone repair system. Split-dose radiationexperiments, that is 8 Gy versus 4 Gy + 4 Gy separated by 3h, were performed to evaluate and contrast the relative abilitiesof CHX and WR-1065, each alone or in combination, in affectingcell survival. Cycloheximide administered to cells 30 min beforethe first radiation dose and present throughout the 3 h incubationtime prior to the second dose inhibited split-dose repair asevidenced by a reduction in surviving fraction by 60% as comparedwith the value obtained for non-CHX-treated cells that wereexposed to two equal doses of 4 Gy. Cells exposed to 4 mM WR-1065immediately following the first 4 Gy radiation dose and thenwashed free 2.5 h before exposure to a second 4 Gy dose, whichwas also followed by a 30 min exposure to WR-1065, increasedthe surviving fraction by 80% over the value obtained for cellsnot exposed to WR-1065 during their split-dose radiation treatment.When CHX treatment was combined with WR-1065 exposure, the protectiveeffect of WR-1065 was abolished, that is surviving cell fractionwas again reduced by 60% as compared with untreated controlgroups. These results indicate that protein synthesis is requiredfor WR-1065 to affect split-dose related repair processes. Presumably,the inhibition of the induction of an error-prone repair systemby CHX would account for its effects on both resultant decreasesin mutation frequency and cell survival. In contrast, WR-1065and/or its disulfide metabolite appear to facilitate the efficacyand fidelity of such a repair system once it is induced. Thisis most probably the result of their polyamine-like structuresand properties, which can influence the stability of DNA damagedsites as well as inhibit cell cycle progression, thus leadingto longer times available in which to complete repair beforedamage is fixed at cell division.     

Relevance of density, size and DNA content of tumour cells to the lung colony assay

Mouse fibrosarcoma tumours were dissociated and divided into subpopulations of viable cells by centrifugation in linear density gradients of Renografin. Two of these subpopulations, designated Band 2 and Band 4, differed in their clonogenic ability in lung colony assay. The less dense Band 2 cells were significantly more clonogenic than the Band 4 cells (2.9 percent vs 1.4 percent respectively). Each band was further separated on the basis of cell size by centrifugal elutriation. Each size class of cells comprising Band 2 showed higher clonogenic ability than the corresponding size class in Band 4. Thus cell size differences were not responsible for the clonogenic differences between these bands. To determine whether cell-cycle distribution of the tumour cells was responsible for differences in cloning efficiency, flow microfluorometric and premature chromosome condensation methods were utilized. The unseparated and Band 4 populations showed a higher percentage of cells in S and G2 than did the Band 2 populations, but many of the S and G2 tumour cells showed extensive chromosome damage. From this study we conclude that the increased clonogenic ability of the lighter tumour cells is not due to differences in cell size or cell-cycle parameters.     

Phase-specific cytotoxicity in vivo of hydroxyurea on murine fibrosarcoma pulmonary nodules.

The cytotoxic effects in vivo of hydroxyurea (HU) on murine fibrosarcoma (FSa) cells grown as pulmonary tumours were determined. Tumour cells from 13-day-old nodules were made into suspension and separated on the basis of cell size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, as well as the degree of contamination by normal diploid cells in each of the tumour-cell populations. HU cytotoxicity was tested by administering both a single 1 mg/g i.p. dose into mice that had been injected i.v. 20 min earlier with known numbers of synchronized viable FSa cells, and i.p. doses of 1 mg/g each into mice bearing 13-day-old pulmonary nodules. In the latter experiments, animals were killed 1 h after the last dose, and the tumour nodules were excised and made into a single-cell suspension and elutriated. Known numbers of cells from each fraction were injected into recipient mice to determine survival. In both sets of experiments, cell killing by HU correlated with the percentage of S-phase cells. The treatment of 13-day-old pulmonary nodules with 3 doses of HU also depleted the (G2+M) phase tumour cells and increased the heterogeneity between tumour subpopulations, as determined by FMF analysis.     

Cytotoxic effect of adriamycin in vivo on synchronized murine fibrosarcoma cells

The cytotoxic effect of Adriamycin (ADR) on synchronized fibrosarcoma (FSa) cells lodged in the lungs of specific pathogen free C₃Hf/Bu mice was determined. FSa cells from primary asynchronous cultures were separated and synchronized on the basis of cell size by centrifugal elutriation. Cell cycle parameters were determined by flow micro-fluorometry (FMF). Viable tumor cells from each elutriator fraction were injected intravenously into recipient mice. Twenty minutes later, ADR (10 mg/kg) was administered intravenously into half of the animals in each group. After fourteen days the animals were killed, and the resulting lung colonies were counted. Under these conditions, the S-phase enriched populations were found to be the most sensitive to ADR. Cell survival ranged from a maximum of 8% to a minimum of 0.3% for the Gi and S-phase enriched populations, respectively. Dose response curves were also determined for selected populations treated in vivo. While no great difference in Do was observed between the survival curves representing each of these populations (range, 0.65–0.71 mg/kg), the extrapolation numbers varied from 0.41 (51% S-phase) to 2.1 (9% S-phase).     

Cytoprotection by WR-1065, the active form of amifostine,is independent of p53 status in human malignant glioma cell lines

Purpose : This study tests the hypothesis that p53 status, i.e. wild type versus mutant form, is a determinant in radiation protection of human glioma cells by WR-1065, the active thiol form of amifostine (WR-2721). Materials and methods : The cytoprotective effectiveness of WR-1065 when present during irradiation was investigated using four well-characterized human glioma cell lines. The p53 positive lines were U87 and D54, and the mutant p53 lines were U251 (mutant at codon 273; CGT/CAT; Arg/His) and A172 (mutant at codon 242; TGC/TTC; Cys/Phe). Treatment conditions included exposure of cells to a range of doses (0–10Gy) alone or in combination with 4 mM of WR-1065 added 30min prior to irradiation. Resultant survival curves were obtained using a clonogenic assay and protection factors, the ratio of terminal slopes +/- WR-1065, were determined for each glioma cell line. Results : The D 0 values of wild-type U87 and D54 were 1.62 and 1.89 Gy while those of p53 mutants U251 and A172 were 1.64 and 1.68 Gy, respectively. Protection factors were determined to be 2.4 and 1.9 for U87 and D54, and 2.6 and 2.8 for U251 and A172, respectively. Conclusions : The p53 status of the four human glioma cell lines tested was not a predictor for either their relative sensitivity to ionizing radiation or ability to be protected by WR-1065. It is concluded that cytoprotection exhibited by cells exposed to WR-1065 during irradiation is independent of their p53 status.     

Protective effects of 2-[(aminopropyl)amino] ethanethiol against bleomycin and nitrogen mustard-induced mutagenicity in V79 cells

This study examines the effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on bleomycin (BLM) and nitrogen mustard- (HN2) induced cytotoxicity, DNA damage, and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells. The anti-mutagenic effect of WR-1065 on cis-diamminedichloroplatinum (cis-DDP) and radiation- (XRT) induced HGPRT mutations was also evaluated for comparative purposes. WR-1065 (4 mM) was added prior to exposure of cells to therapy agents. All exposure times were 30 min. and both cell survival and mutagenesis were assayed. WR-1065 was effective in protecting against both effects. The induction of mutants corrected for background by BLM, HN2, cis-DDP, or XRT was linear in all cases. Mutation frequencies without WR-1065 were 78 X 10(-6) per unit BLM, 66 X 10(-7) per microgram HN2, 25 X 10(-7) per microgram cis-DDP; and 87 X 10(-7) per Gy of XRT. With WR-1065, these were reduced to 37 X 10(-6) per unit BLM, 40 X 10(-7) per microgram HN2, 1 X 10(-7) per microgram cis-DDP, and 44 X 10(-7) per Gy of XRT. Mutation protection factors (MPF), a ratio of the corresponding slopes of the mutation induction curves, with and without WR-1065 were: BLM, MPF = 2.8; HN2, MPF = 3.4; cis-DDP, MPF = 7.1; and XRT, MPF = 5.1. Single-strand-break (SSB) formation in DNA by BLM or HN2, assayed by alkaline elution, was protected against by WR-1065. WR-1065 did not induce SSB in control cells. The reduction of the mutagenic effects of agents used in radiation and chemotherapy by radioprotectors may be an important additional benefit for consideration in their use in cancer treatment.