Effect of tumor dissaggregation on results of in vitro cell survival assay after in vivo treatment of the EMT-6 tumor: x-rays, cyclophosphamide, and bleomycin

EMT-6 tumors were treated in vivo with 300 kVp X-rays, cyclophosphamide, or bleomycin. Tumor cell suspensions were prepared by digesting tumors with trypsin or a collagenase-deoxyribonuclease-pronase cocktail, and cells were plated in vitro for determination of fractional cell survival. Cell survival after X-rays was identical for the two disaggregation methods. Trypsin-derived cells were far more sensitive to bleomycin but less sensitive to cyclophosphamide than those prepared with the mixed enzyme cocktail. Interaction of drug produced and enzyme caused damage was the probable cause for these discrepancies. The nature of the interaction may be drug specific and therefore unpredictable. The results were unlikely to be due to different nonrepresentative tumor cell samples being produced by the two digestion methods, because the X-ray cell survival curves were so similar for the two products.     

Degradation of DNA and structure-activity relationship between bleomycins A2 and B2 in the absence of DNA repair.

The contribution of DNA repair to the net number of DNA breaks produced during chemical degradation of DNA was determined by using temperature-sensitive mutant cells deficient in ATP-dependent DNA ligase [poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase, EC 6.5.1.1]. In a very sensitive assay for determining lesions introduced into Saccharomyces cerevisiae DNAs, 2-14C- and 6-3H-prelabeled DNAs from ligase-proficient and ligase-deficient cells were sedimented together through precalibrated, isokinetic alkaline sucrose gradients. DNA ligation was slower after chemical degradation of DNA by bleomycin than after gamma irradiation. DNA breaks increased approximately linearly with drug concentrations, and were approximately equivalent for ligase-proficient and ligase-deficient cells. These results were unexpected because ligase-deficient, but not ligase-proficient, cells lacked the capacity to eliminate DNA breaks produced by bleomycin. The results indicated that DNA repair did not occur during the chemical degradation of DNA under the experimental conditions. Bleomycin B2 produced considerably more DNA breaks than bleomycin A2 over a range of concentrations in ligase-proficient cells, which tolerated higher numbers of DNA breaks in general than ligase-deficient cells. The chemical analogues are structurally identical except for their cationic C-terminal amine. The actual number of DNA breaks produced by bleomycin A2 or bleomycin B2, and not the concentration of bleomycin A2 or bleomycin B2 per se, determined the amount of cell killing. DNA repair is critical in quantitating DNA breaks produced by chemicals, but was ruled out as a factor in the higher DNA breakage by bleomycin B2 than bleomycin A2.     

Functional EpoR Pathway Utilization Is Not Detected in Primary Tumor Cells Isolated from Human Breast,Non-Small Cell Lung,Colorectal, and Ovarian Tumor Tissues

Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.     

Cellular resistance to bleomycin in Saccharomyces cerevisiae is not affected by changes in bleomycin hydrolase levels.

Bleomycin is a glycopeptide drug that exerts potent genotoxic potential and is highly effective in the treatment of certain cancers when used in combination therapy. Unfortunately, however, tumors often develop resistance against bleomycin, and the mechanism of this resistance remains unclear. It has been postulated that bleomycin hydrolase, a protease encoded by the BLH1 gene in humans, may account for tumor resistance to bleomycin. In support of such a notion, earlier studies showed that exogenous expression of yeast Blh1 in human cells can enhance resistance to bleomycin. Here we show that (i) yeast blh1delta mutants are not sensitive to bleomycin, (ii) bleomycin-hypersensitive yeast mutants were no more sensitive to this agent upon deletion of the BLH1/LAP3/GAL6 gene, and (iii) overproduction of Blhl in either the parent or bleomycin-hypersensitive mutants did not confer additional resistance to these strains. Therefore, yeast Blh1 apparently has no direct role in protecting this organism from the lethal effects of bleomycin, even though the enzyme can degrade the drug in vitro. Clearly, additional studies are required to establish the actual biological role of Blh1 in yeast.     

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. III. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by bleomycin, mono- and bi-functional alkylating agents

Two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6, were characterised with regard to their responses to genotoxic chemicals, namely bleomycin, MMS, EMS, MMC and DEB for induction of cell killing, chromosomal aberrations and SCEs at different stages of the cell cycle. In addition, induction of mutations at the HPRT and Na+/K+ ATPase (Oua) loci was evaluated after treatment with X-rays and MMS. Xrs 5 and xrs 6 cells were more sensitive than wild-type CHO-K1 to the cell killing effect of bleomycin (3 and 13 times respectively) and for induction of chromosomal aberrations (3 and 4.5 times). In these mutants a higher sensitivity for induction of chromosomal aberrations to MMS, EMS, MMC and DEB was observed (1.5-3.5 times). The mutants also showed increased sensitivity for cell killing effects of mono- and bi-functional alkylating agents (1.7-2.5 times). The high cell killing effect of X-rays in these mutants was accompanied by a slight increase in the frequency of HPRT mutation. The xrs mutants were also more sensitive to MMS for the increased frequency of TGr and Ouar mutants when compared to wild-type CHO-K1 cells. Though bleomycin is known to be a poor inducer of SCEs, an increase in the frequency of SCEs in xrs 6 cells (doubling at 1.2 micrograms/ml) was found in comparison to no significant increase in xrs 5 or CHO-K1 cells. The induced frequency of SCEs in all cell types increased in a similar way after the treatment with mono- or bi-functional alkylating agents. MMS treatment of G2-phase cells yielded a higher frequency of chromatid breaks in the mutants in a dose-dependent manner compared to no effect in wild-type CHO-K1 cells. Treatment of synchronised mutant cells at G1 stage with bleomycin resulted in both chromosome- and chromatid-type aberrations (similar to the response to X-ray treatment) in contrast to the induction of only chromosome-type aberrations in wild-type CHO-K1 cells. The frequency of chromosomal aberrations chromosome and chromatid types) also increased with MMC treatment in G1 cells of xrs mutants. DEB treatment of G1 cells induced mainly chromatid-type aberrations in all cell types. The possible reasons for the increased sensitivity of xrs mutants to the chemical mutagens studied are discussed and the results are compared to cells derived from radiosensitive ataxia telangiectasia patients.     

Modification of lymphokine-activated killer cell accumulation into tumor sites by chemotherapy, local irradiation, or splenectomy

The effects of chemotherapy or local irradiation on lymphokine-activated killer (LAK) cell accumulation into tumor sites were investigated. Lymphokine-activated killer cells labeled with 111In-oxine were injected into the caudal vein of C57BL/6 mice that had been previously transplanted with 3LL cancer. An adoptive transfer of LAK cells was carried out 4 days after treatments. Twenty-four hours after the transfer, tumor tissues were excised, and the accumulation of labeled LAK cells in the tumor was measured. In two different experiments, LAK cell accumulation in tumor in the nontreated group was 2.15% and 1.58% of the administered dose per gram of tissue. The accumulation in the groups of mice treated with cyclophosphamide, nimustine hydrochloride, or Adriamycin increased fourfold (7.38% dose/g, 6.61% dose/g), threefold (6.47% dose/g) and twofold (4.46% dose/g), respectively, as compared with the nontreated group. These agents induced significant tumor regression. In the group treated with bleomycin, which showed no significant effect on tumor growth, LAK cell accumulation in tumor remained unaltered (1.57% dose/g). However, the group treated with local irradiation, which induced significant tumor reduction, showed no increase in LAK cell accumulation into tumors. These results suggest that some antitumor drugs enhance LAK cell accumulation into tumor sites and that this increase is due to tumor modification by antitumor drugs.     

Combined ex vivo treatment with immunotoxins and mafosfamid: a novel immunochemotherapeutic approach for elimination of neoplastic T cells from autologous marrow grafts

We evaluated a novel ex vivo "purging" protocol for selective elimination of neoplastic T cells from human marrow by using a sensitive clonogenic assay. Immunotoxins (IT) were synthesized by conjugating ricin (R) to four different monoclonal antibodies (MoAb) directed against distinct markers of T cell lineage. Treatment with anti-p67-R produced effective elimination of leukemic T cells from human marrow. The cyclophosphamide congener mafosfamid (ASTA Z 7577) markedly enhanced the target cell cytotoxicity of IT and extended the final level of clonogenic kill 2 to 3 logs. Our data show that anti-p67-R in combination with mafosfamid resulted in a maximum elimination of 6.2 logs of neoplastic T cells with minimal toxicity to normal bone marrow progenitors. The efficiency of this protocol was not reduced in the presence of excess normal bone marrow cells. Similar findings were obtained by using a cocktail of four different anti-T cell IT. This approach is unique in combining both immunologic (IT) and chemical (mafosfamid) strategies for more effective ex vivo bone marrow purging in autologous bone marrow transplantation for T cell acute lymphoblastic leukemia/lymphoblastic lymphoma.     

Stromelysin-3 suppresses tumor cell apoptosis in a murine model

Stromelysin-3 (STR-3) is a matrix metalloproteinase with a unique pattern of expression and substrate specificity. During embryogenesis and remodeling of normal adult tissues, STR-3 is produced by stromal cells in direct contact with epithelial cells undergoing regional apoptosis and selective cell survival. STR-3 is also overexpressed by interdigitating stromal cells in primary epithelial malignancies. Although STR-3 does not degrade classic extracellular matrix components, the enzyme promotes the establishment of local tumors in nude mice by as yet undefined mechanisms. STR-3 is induced when malignant epithelial cells come into contact with surrounding stromal elements; the active stromal cell-derived 45 kDa enzyme is subsequently processed to a 35 kDa protein without enzymatic activity. We have generated MCF-7 transfectants expressing wild type or catalytically inactive 45 kDa STR-3 (STR-3wt and STR-3cat-) or secreted 35 kDa STR-3 (35 kDa STR-3sec) and evaluated their implantation and survival in nude mice. Tumors developed significantly more rapidly in animals receiving STR-3wt, rather than vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Most importantly, STR-3wt tumors had a significantly lower percentage of apoptotic cells than tumors derived from vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Taken together, these studies suggest that the active STR-3 enzyme may increase tumor take by suppressing tumor cell apoptosis and that 45 kDa to 35 kDa STR-3 processing limits STR-3 activity at the tumor/stromal interface. Because STR-3 is secreted as an active enzyme rather than a proform, subsequent 45 kDa to 35 kDa STR-3 processing may represent a novel mechanism for regulating enzymatic activity.     

Apoptotic response of malignant rhabdoid tumor cells

BACKGROUND: Malignant rhabdoid tumors (MRTs) are extremely aggressive and resist current radio- and chemotherapic treatments. To gain insight into the dysfunctions of MRT cells, the apoptotic response of a model cell line, MON, was analyzed after exposure to several genotoxic and non-genotoxic agents employed separately or in association. RESULTS: Fluorescence microscopy of chromatin morphology and electrophoretic analysis of internucleosomal DNA fragmentation revealed that MON cells were, comparatively to HeLa cells, resistant to apoptosis after treatment with etoposide, cisplatin (CisPt) or X-rays, but underwent some degree of apoptosis after ultraviolet (UV) C irradiation. Concomitant treatment of MON cells with X-rays or vinblastine and the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin resulted in synergistic induction of apoptosis. Western blot analysis showed that the p53 protein was upregulated in MON cells after exposure to all the different agents tested, singly or in combination. In treated cells, the p53 downstream effectors p21WAF1/CIP1, Mdm2 and Bax were induced with some inconsistency with regard to the accumulation of p53. Poly ADP-ribose polymerase (PARP) cleavage, indicative of ongoing apoptosis, occurred in UVC-irradiated cells and, especially, in cells treated with combinations of X-rays or vinblastine with wortmannin. However, there was moderate or no PARP cleavage in cells treated with CisPt, X-rays, vinblastine or wortmannin singly or with the combinations X-rays plus CisPt or vinblastine and CisPt plus vinblastine or wortmannin. The synergistic effect on the induction of apoptosis exerted by some agent combinations corresponded with synergy in respect of MON cell growth inhibition. CONCLUSION: These results suggest abnormalities in the p53 pathway and apoptosis control in MRT cells. The Ras/PI3-K/AKT signaling pathway might also be deregulated in these cells by generating an excess of survival factors. These dysfunctions might contribute to the resistance of MRTs to current antineoplastic treatments and could warrant consideration in the search of new therapeutic approaches.     

The in vitro and in vivo antitumor effects of pepleomycin alone or in combination with radiation

Pepleomycin is a derivative of bleomycin, which is less toxic to the host but possesses greater antitumor activity. Experiments are described here in which Chinese hamster V-79 cells in culture and a murine squamous cell carcinoma in vivo have been used to obtain survival curves for pepleomycin alone or in combination with radiation. In both systems the survival curves for pepleomycin alone are biphasic, and this drug proved to be twice as effective as bleomycin. Further, in contrast to bleomycin, which showed simple additivity with radiation, pepleomycin potentiated radiation injury to the tumor cells both in vivo and in vitro. Therefore, pepleomycin may be superior to bleomycin not only in drug therapy but also in combined modality therapy.     

Host cell cytotoxicity, cellular repopulation dynamics, and phase-specific cell survival in X-irradiated rat rhabdomyosarcoma tumors

Postirradiation tumor volume response, cellular repopulation dynamics, cell-cycle perturbations, and phase-specific cell survival were characterized in rat rhabdomyosarcoma R-1 tumors (the R2C5 subline) following an in situ 10-Gy dose of 225-kVp X rays. This X-ray dose produced a 7.5-day delay in tumor growth to twice the volume measured at the time of irradiation, and reduced the initial surviving fraction of R2C5 cells to 0.17 as measured by the excision assay procedure. The surviving fraction of R2C5 cells returned to unity by the 16th day after tumor irradiation. On the basis of flow cytometry measurements of DNA content in tumor cells stained with a noncytotoxic concentration of Hoechst 33342 (5 microM, 2 h, 37 degrees C), a transient G2 block was observed 1 day after irradiation. Flow cytometry measurements also demonstrated that the tetraploid R2C5 cells constituted only 30% of the total tumor cell population, with the remainder being diploid host cells comprised of macrophages, monocytes, lymphocytes, and granulocytes. Large numbers of host cells infiltrated the irradiated tumors, leading to an increase in the percentage of diploid cells by Day 2 and reaching a level of more than 80% of the total tumor cell population by 4 to 8 days after irradiation. The influx of host cells into irradiated tumors was correlated temporally with a significant 12-fold decrease in the surviving fraction of R2C5 cells that occurred between Days 2 and 4 postirradiation. When the diploid host cell population was removed by cell sorting procedures, the surviving fraction of R2C5 cells at Day 4 was substantially greater than that in the presence of the host cells. Experiments involving the mixing of 4/1 and 12/1 ratios of diploid host cells and tetraploid tumor cells isolated from irradiated and unirradiated tumors demonstrated that the cytotoxic effect of the host cells was specific for the irradiated tumor cells. The significant toxic effect of host cells on irradiated tumor cells was observed only at 2 to 4 days after irradiation, and not at earlier or later times. The data obtained in these experiments indicate that the immunogenicity of R2C5 cells is increased significantly by irradiation, and a resultant cell-mediated host immune response produced a delayed decrease in tumor cell survival that is most pronounced 4 days after irradiation. The cell survival characteristics of R2C5 cells in different cell-cycle phases were found to be similar through the 16-day postirradiation interval that was studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

A murine plasmacytoma MOPC 104E resistant to cyclophosphamide is resistant to immunotherapy

Summary A murine plasmacytoma MOPC 104E (MOPC) is highly sensitive to chemotherapeutic agents such as cyclophosphamide and mitomycin C as well as to immunotherapy (OK-432-combined adoptive immunotherapy using interleukin-2-cultured killer cells). In the present study, we prepared cyclophosphamide-resistant MOPC cells (MOPC-CPA/R) by serial in vivo passage of tumor cells following cyclophosphamide treatment. The in vivo sensitivity of MOPC-CPA/R to mitomycin C or to immunotherapy (OK-432-combined adoptive immunotherapy) was significantly decreased compared to the parent MOPC. In vitro experiments showed that MOPC-CPA/R were more resistant (five-fold) to lysis by cultured immune spleen cells than MOPC. Inhibition of the lytic activity of cultured immune spleen cells against MOPC was significantly increased (P <0.05) by the addition of unlabeled MOPC compared to unlabeled MOPC-CPA/R. These results suggest that MOPC-CPA/R express weaker antigenicity than MOPC. However, the transfer of immune spleen cells cultured with tumor extract derived from MOPC-CPA/R significantly prolonged the survival of MOPC-CPA/R-inoculated mice. Thus, by repeated cyclophosphamide treatment, tumor cells with low-antigenicity were selected. These tumor cells had lower sensitivity to another chemotherapeutic agent and immunotherapy. Such an immunological response may play an important role in cancer therapy.     

Intracellular degradation of bleomycin hydrolase in two Chinese hamster cell lines in relation to their peplomycin susceptibility

The Chinese hamster lung (V79) cell was intrinsically 10-times more resistant to peplomycin, a bleomycin-related antitumor antibiotic, than the Chinese hamster ovary (CHO) cell. This may be associated with the 3-times higher levels of recovery of bleomycin hydrolase activity of the V79 cell. The degradation of bleomycin hydrolase molecules in both V79 and CHO cells was examined using a monoclonal antibody specific for the enzyme. Labelling experiments showed that the bleomycin hydrolase in CHO cells was less stable than the comparable enzyme in V79 cells, and that 48 kDa subunits comprising bleomycin hydrolase (a homohexameric enzyme) molecules were degraded into 31 kDa forms in both cell lines. The 105,000 X g pellet (microsomes) fraction obtained after subcellular fractionation of CHO cells contained both 48 kDa subunit and 31 kDa forms of bleomycin hydrolase, while the 105,000 X g supernatant cytosol fraction yielded only 48 kDa subunit forms of the enzyme. Moreover, bleomycin hydrolase activity of both V79 and CHO cells was almost entirely recovered from the cytosol fraction. These results suggest that degradation of the 48 kDa subunit form of bleomycin hydrolase in these two lines of cultured cells into the 31 kDa form occurs on the plasma membrane or the endoplasmic reticulum, with which the resulting large number of bleomycin hydrolase molecules or degraded forms of the enzyme that have lost enzymatic activity are associated.     

Hypersensitivity to ionizing radiation, in vitro, in a new chromosomal breakage disorder, the Nijmegen Breakage Syndrome

The Nijmegen Breakage Syndrome (NBS) is a new chromosomal instability disorder different from ataxia telangiectasia (AT) and other chromosome-breakage syndromes. Cells from an NBS patient appeared hypersensitive to X-irradiation. X-rays induced significantly more chromosomal damage in NBS lymphocytes and fibroblasts than in normal cells. The difference was most pronounced after irradiation in G2. Further, NBS fibroblasts were more readily killed by X-rays than normal fibroblasts. In addition, the DNA synthesis in NBS cells was more resistant to X-rays and bleomycin than that in normal cells. The reaction of NBS cells to X-rays and bleomycin was similar to that of cells from patients with ataxia telangiectasia. Our results indicate that NBS and AT, which also have similar chromosomal characteristics, must be closely related.     

Chronic intratumoral chemotherapy of a rat tumor with cisplatin and fluorouracil

In order to demonstrate the feasibility and efficacy of intratumoral chemotherapy, brain tumors in rats were treated by direct infusion of cisplatin or fluorouracil. Each animal was initially implanted in the midline cerebellum with a chronic stainless-steel cannula, and 2 weeks later 1 X 10(5) 9L cells were injected through the cannula. 8 days after tumor cell transplantation, a small implantable pump containing drug, or saline as a control, was connected up to the same cannula, and the solution was pumped into the tumor region for 7 days. The results showed that both drugs produced statistically significant increases in survival as compared to the controls.     

Assessment of electroporation by flow cytometry

BACKGROUND: Electroporation accomplishes transient permeabilization of cells and thus aids in the uptake of drugs. The method has been employed clinically in the treatment of dermatological tumors with bleomycin. The conditions of electroporation are still largely empirical and information is lacking as to the interrelationships among voltage pulse height, pulse number and toxicity, cell permeation, drug uptake, and effects on drug toxicity. We used propidium iodide (PI) and flow cytometry to define cell permeation into cytoplasmic and nuclear compartments to determine the improvements of drug toxicity that can be accomplished by electroporation. METHODS: Human squamous carcinoma cells of defined TP53 status and normal human epithelial cells were subjected to electroporation using a square wave pulse generator in the range of 0-5,000 V/cm. Flow cytometry served to establish entry of the drug reporter, PI, into the cytoplasm and nucleus. A dye staining method served to establish cell survival and to determine the toxicity of bleomycin alone, electroporation alone, and electroporation with bleomycin. RESULTS: The electric field intensity (EFI) required to produce 50% permeabilization (EP(50)) is cell type dependent. The EP(50) varied from 1,465 to 2,027 V/cm. An EFI below 900 V/cm is growth stimulatory whereas an EFI in excess of 1,000 V/cm is growth inhibitory. An EFI of 1,000 V/cm is sufficient to increase bleomycin toxicity by a factor of 2-3. A differential electroporation efficiency is observed between normal and tumor cells. CONCLUSIONS: Tumor cells can be targeted preferentially at electroporation voltages where normal cells are less permeable.     

Establishment and characterization of EB virus-free normal B-lymphocyte and interleukin-6-producing poorly differentiated adenocarcinoma cell lines derived from gastric tumor tissue

We successfully established two cell lines, an adenocarcinoma cell line (designated as HIGS) and Epstein-Barr virus-free normal B-lymphocyte cell line (designated as HIGS-BL), derived from a moderately to poorly differentiated adenocarcinoma of the stomach, and examined their characteristics. The tumor delivered to our laboratory from an operating room was cut into small pieces and cultured on the dishes. HIGS and HIGS-BL were established from each individual dish after the onset of primary culture. Although their culture methods were the same, the HIGS cell line was not established from the dishes growing HIGS-BL cells. In addition, HIGS-BL cells were scarcely observed in the HIGS cell dishes. Because of these factors, we have considered until now that HIGS-BL cells may inhibit the growth of HIGS cells or cause damage to HIGS cells by unknown mechanisms. Injection of HIGS-BL cells, other B-lymphocyte cell lines, or the conditioned media of HIGS-BL cells into nude mice bearing HIGS-grafted tumors was performed individually. When HIGS and HIGS-BL cells were co-cultured in the same dishes, HIGS-BL cells inhibited the proliferation of HIGS cells. The inhibition of grafted tumor growth was confirmed by the injection of not only the HIGS-BL cells but also the B-lymphocytes. Furthermore, this inhibition was only observed when the conditioned medium of B-lymphocytes was injected into the nude mice. These results suggested that the secretory products by general B-lymphocytes (including HIGS-BL) have some ability to inhibit the proliferation of HIGS cells. In addition, susceptibility tests to anti-cancer drugs suggested that HIGS cells were sensitive to CDDP, ADM and MMC, and HIGS-BL cells were sensitive to CDDP. If CDDP was used for chemotherapy in the patient, the drug produced atrophy of HIGS-BL cells. The study about HIGS and HIGS-BL cells reported the necessity for novel therapeutic approaches in oncotherapy.     

Enhancing tumor implantation and growth rate of Ramos B-cell lymphoma in nude mice

The ability of a human B-cell lymphoma cell line to grow subcutaneously as tumors in nude mice was investigated. The effect of pretreating mice with cyclophosphamide or whole-body irradiation (WBI) was compared with no pretreatment of the mice. Both methods of pretreatment resulted in a higher tumor implantation rate, compared with that for non-pretreated controls. In mice that underwent WBI-pretreatment, a tumor implantation rate of 100% was observed, whereas mice pretreated with cyclophosphamide had a tumor implantation rate of 80%. In non-pretreated control mice, an implantation rate of only 50% was observed. Three weeks after injection, tumor size was significantly larger in mice of the pretreated groups, compared with that in mice of the group that did not receive pretreatment. Furthermore, particularly in the group pretreated with WBI, the tumors grew more synchronously, compared with tumors in the control group. Results of this study indicate that pretreatment with cyclophosphamide or WBI improves the tumor implantation rate of Ramos cells in nude mice, providing a workable animal model for studying human B-cell lymphoma.     

The relationship between chromosomal aberrations, survival and DNA repair in tumour cell lines of differential sensitivity to X-rays and sulphur mustard

A pair of cultured rat lymphosarcoma cell lines (Yoshida) with a pronounced differential sensitivity to killing with sulphur mustard (SM), but with the same sensitivity to X-rays, was examined for chromosome damage and DNA repair replication after treatment with these agents. A pair of mouse lymphoma cell lines (L5178Y) with a differential sensitivity to X-rays was similarly investigated.SM-resistant Yoshida cells suffered much less chromosome damage than sensitive cells in spite of equal alkylation of DNA, RNA and protein in sensitive and resistant cells. The pair of Yoshida cell lines sustained the same amount of chromosome damage after X-irradiation. Much less chromosome damage was observed in the radiation-resistant lymphoma cell line than in the sensitive line after X-irradiation.No differences was found between the pairs of cell lines in their capacities for repair replication after SM or X-ray treatment.Thus, the drug and radiation resistance is accompanied by, and perhaps mediated through, a reduced amount of induced chromosome damage but is not quantitatively related to the capacity for DNA repair replication.Apart from small differences in modal chromosome numbers there are no obvious karyotype differences between the sulphur mustard-sensitive and -resistant Yoshida cells or between the radiation-sensitive and -resistant lymphoma cells.     

The immunogenic properties of drug-resistant murine tumor cells do not correlate with expression of the MDR phenotype

Alterations in the immunogenic properties of tumor cells frequently accompany selection for multipledrug-resistant (MDR) variants. Therefore, studies were performed to examine the hypothesis that overexpression of membrane P-glycoprotein, commonly observed in MDR tumor cells, is associated with enhanced immunogenic properties. Immunogenicity was determined by (a) the ability of drug-sensitive parental UV2237M fibrosarcoma cells and drug-resistant UV2237M variant cells to immunize normal mice against rechallenge with parental tumor cells and (b) the ability of normal syngeneic mice to reject cell inocula that caused progressive tumor growth in immunocompromised mice. Variant UV2237M cell lines included subpopulations selected for a six- to ten-fold increase in mRNA for P-glycoprotein and expression of the MDR phenotype (resistance to doxorubicin) and cells sensitive to doxorubicin (and no expression of MDR properties) but resistant to ouabain. All UV2237M drug-resistant cells were highly immunogenic in immunocompetent mice, regardless of their MDR phenotype. Additional studies showed that CT-26 murine adenocarcinoma cells, sensitive or resistant to doxorubicin (expressing high levels of P-glycoprotein), injected into normal syngeneic Balb/c mice produced rapidly growing tumors. The data do not demonstrate a correlation between the immunogenic properties of drug-resistant tumor cells and the expression of P-glycoprotein.Supported in part by core grant CA-16672 R35-CA42 107 from the National Cancer Institute, and postdoctoral fellowship grant PF-3446 from the American Cancer Society (R. R.)