Response of lymphosarcoma LS/BL cells to continuous irradiation.

Mouse lymphosarcoma LS/BL cells growing as an ascites tumor in the peritoneal cavity of C57BL mice were continuously irradiated in vivo at a low exposure rate of 1.2 Gy per day (5 rad/hr). The growth of the ascites tumor evaluated by direct counting of the cells in the peritoneal cavity and their capacity to form colonies in livers declined with increasing time of continuous irradiation. The radiosensitivity and repair ability of LS/BL cells were studied by a serial dilution method using host survival time as the end point and by the liver colony assay. The radiosensitivity of continuously irradiated LS/BL-CI cells showed no remarkable change as measured by the Do values, but from the 150th week of irradiation the initial shoulder on the survival curves appeared and its width increased with time of exposure. The extrapolation number (n) increased from 1.0 to 8.4 after 350 weeks of irradiation. The reappearance of the initial shoulder was proved with the split-dose technique.

The response of mouse lymphosarcoma cells to continuous gamma irradiation.

Long-lasting survival of LS/BL tumor cells under continuous exposure in vivo to 60Co irradiation at an exposure rate of 10 cGy/h was observed. While LS/BL tumor cells tolerated the irradiation for more than 150 weeks, the number of hematopoietic stem cells decreased to less than 1% of control values within 7 days of irradiation. The liver colony method was used to develop sublines from nonirradiated and continuously (110 weeks) irradiated LS/BL cells for the analysis of karyotypes of the resulting clones of lymphosarcoma cells. The appearance of new structural abnormalities t(12:9) in one of the sublines tested correlated with its faster host killing when compared to the parent line.

Interferon inducer, polyriboguanylic.polyribocytidylic acid, inhibits experimental hepatic metastases in mice.

The antitumour activity of an interferon inducer, the double-stranded complex of polyriboguanylic.polyribocytidylic acid was studied on murine lymphosarcoma LS/BL. The antitumour effect was determined with the aid of an experimental liver-colony model and compared to that exhibited by another synthetic RNA, polyriboinosinic.polyribocytidylic acid. We found that both polynucleotide complexes decreased the number of liver colonies and prolonged the survival of the tumour-bearing mice. This effect was only observed if the complexes were applied in an appropriate dose schedule which included the administration of the drug prior to tumour cell inoculation and subsequent continuous treatment. We have also verified that the polynucleotide complexes did not exert their antitumour effect by a direct action on tumour cells.

The cytotoxic effect of methotrexate as evaluated by colony-forming activity of hemopoietic and tumor cells.

The response of hemopoietic and/or lymphosarcoma cells to methotrexate has been investigated. The proliferative capacity of hemopoietic tissue was measured by exogenous CFU assay. It was inhibited by a six times lower amount of MTX than the proliferative activity of lymphosarcoma cells as evaluated by liver-colony assay. The natural resistance of LS/BL lymphosarcoma cells to MTX might be attributed to the DHFR gene amplification resulting from DNA rearrangements appearing during malignant transformation.

Consistent chromosome abnormalities in LS/BL murine lymphosarcoma cells.

The LS/BL lymphosarcoma was induced by radiation in a C57BL/10 mouse in 1963 and was converted to ascites form in the first mouse transfer generation. In the course of cultivation in vivo the modal number of chromosomes dropped from the initial value 42-41 to 39 (73%). Cytogenetic characterization of the LS/BL tumor was carried out, using G-banding technique. Chromosome abnormalities were consistent in the cell line and involved chromosomes Nos. 3, 6, 12, 13, 16, and X. The most frequent abnormality was the presence of three markers and trisomy of chromosome No. 16.

The growth and dissemination of continuously irradiated LS/B1 lymphosarcoma cells.

Transplantable strain of murine lymphosarcoma growing as a free suspension in the peritoneal cavity of isologous hosts was continuously irradiated by 60Co unit at a low exposure rate of 1.2 Gy/day for 350 weeks. Long-term irradiation did not alter substantially the viability and invasiveness of tumor cells. In the host of irradiated cells the spleen mass was six times higher around the day 8. Histological sections of the spleen revealed areas of edema and hemorrhage and infiltration and proliferation of tumor cells mostly in subcapsular area. Most of the splenomegaly is considered to be due to hyperemia caused by mass of erythrocytes in dilated spleen sinuses. Continuous irradiation is supposed to lead to more pronounced sarcomatous character of LS/B1 tumor.

Antimetastatic action of diltiazem on LS/BL tumor cells in liver tumor-colony assay.

Inhibition of experimental metastases of lymphosarcoma LS/BL cells by diltiazem, a calcium blocking agent, was tested. Diltiazem treatment (2 X 30 mg/kg p.o. for 12 days) resulted in a maximum of 72.8% inhibition of liver metastases. The authors suggest that diltiazem might become suitable candidate for antimetastatic treatment in humans.

Evaluation of cytotoxic and antitumor effects of a tetravalent analog of carboplatin.

The cytostatic and cytocidal effects of a newly synthesized tetravalent platinum drug (CBDCA-ox) on two experimental ascites tumors as well as on normal tissues of the mouse were investigated. It was found that CBDCA-ox exhibited only negligible acute toxicity in comparison with its reduced analog. Also hemotoxicity of this tetravalent drug was markedly lower than that of CBDCA. The comparison of antitumor activity of CBDCA and CBDCA-ox tested against LS/BL lymphosarcoma and EAT tumor cells strongly supports the view that CBDCA-ox deserves further preclinical testing.

Variability of chromosomes in the VUP permanent cell line derived from uveal malignant melanoma.

A permanent cell line [VUP] derived 31 years ago from human malignant melanoma of the choroid has been characterized by genetically firmly anchored heteronuclearity. The most significant chromosomal changes of this cell line are: high instability of the chromosome No. 13 with the rise of new chromosomes formed by translocations, homologous stability of chromosomes 6, 15, and X. Structural changes were not revealed in chromosomes 15 and 22. The variability of chromosomes was studied both by classical conventional methods as well as with GTG banding and DNA hybridization in situ (FISH). Structural diversity was demonstrated in a number of morphologically congruent chromosomes. For example, X chromosome classified morphologically as chromosome No. 10 was determined by means of FISH technique, as a centric fragment Xq with translocated acentric fragment of other chromosomes. Furthermore, mar-t, previously considered to be q arm of chromosome No. 4, is formed by a centric fragment of chromosome No. 13 and an acentric fragment of chromosome No. 1.

A morphological study of continuously irradiated lymphosarcoma cells.

Mouse lymphosarcoma LS/BL cells, cultivated as a free suspension in the peritoneal cavity of C57Bl mice, were continuously irradiated by 60Co unit at an exposure rate of 35.8 x 10(-5) mC kg-1 s-1 (5 R/h). After 250 and 350 weeks of irradiation these cells were examined in light and electron microscope. Both the cytoplasm and the nucleus underwent morphological alterations under continuous exposure. Areas of advanced cytoplasmic degradation were often observed and many nuclei showed wide indentations and prolongation of their shape. The number of mitochondria and vacuoles in the cytoplasm was increased markedly in the irradiated cells. The authors consider some of the described alterations caused by continuous irradiation to act as compensatory mechanisms of the impaired cell.

The effect of biopreparation cACPL on the proliferative activity of haemopoietic and tumour cells.

The haemotoxicity and antitumour activity of crude anticancer phospholipid (cACPL) was evaluated. Results of our experiments gave evidence that the cACPL has no ill effects on haemopoietic stem cells but it markedly stimulates their proliferation. There was little if any anticancer activity of cACPL proved in experiments with murine lymphosarcoma LS/BL and carcinoma EAT cells.

Changes in chromosome patterns of murine lymphosarcoma LS/BL cells during long-term continuous irradiation in vivo.

The chromosome pattern of murine lymphosarcoma LS/BL and its changes during continuous exposure to ionizing radiation in vivo were studied. Absorbed dose rates (60Co) were 10 and 50 mGy/h. The hypodiploid sets with 39 chromosomes (73%) predominated in the control, unirradiated line. The continuously irradiated LS/BL-CI line exhibited insignificant changes in the modal chromosome number. The number of structural aberrations and sister chromatid exchanges was increased.

Behaviour of murine lymphosarcoma cells during prolonged cultivation in vivo.

Transplantable strain of murine lymphosarcoma growing as a free suspension in the peritoneal cavity of isologous hosts was isolated after fractionated irradiation of C57BL mice. A 14-year cultivation led to changes in its properties; the mean survival time of the hosts was shortened, the modal number of chromosomes changed from 41--42 to 39 and their radioresistance increased slightly. Strain specificity remained unchanged.

Continuous irradiation of a murine lymphosarcoma in vivo.

Cells of an ascites tumour LS/BL growing in the peritoneal cavity of C57BL mice were continuously irradiated in vivo at a low exposure rate of 3.58 X 10(-7) C.kg-1.s-1 (5R/h) for several years. The total volume of the ascites tumour evaluated by counting cells in the peritoneal cavity declined with increasing time of irradiation. However, the viability of tumour cells was fully retained so that upon an intravenous injection of the same amount of cells they killed their hosts as rapidly as the unirradiated cells. Longer survival was noted in the hosts given the irradiated LS/BL cells subcutaneously. In the hosts of irradiated cells, the spleen weight was greater around day 8 after transfer and the 3H-thymidine incorporation into DNA was increased.

Inhibition of DNA synthesis in mouse lymphosarcoma LS/BL cells by fluorocitrate.

Fluorocitrate is a specific inhibitor of aconitase activity (EC 4.2.1.3). It causes an inhibition of citric acid cycle reactions and consequently reduces the oxygen consumption, as well as the total volume of oxidative phosphorylations. Experiments using 3H-thymidine incorporation into mouse lymphosarcoma LS/BL cells proved that the in vitro blocking of citric acid cycle by fluorocitrate (3.6 and 7.2 mM) was accompanied by a decrease in DNA synthesis by 40 to 60 percent, as compared to control cells. A similar inhibitory effect upon DNA synthesis was also found in cells cultured in the abdominal cavity of host mice injected intraperitoneally with 5 mg./kg. of sodium fluoroacetate. Following the injection, fluorocitrate is formed in vivo by way of Peters' lethal synthesis.

Effect of continuous irradiation upon some biological properties of murine lymphosarcoma LS/BL.

Mouse lymphosarcoma LS/BL cells passaged in syngeneic hosts were continuously irradiated with 60Co gamma rays at an exposure of 1R/hour. Long-term irradiation did not substantially alter the proliferative rate of leukaemic cells. Radiation exposure in the duration of 60 and 70 weeks led to an increase in the mean survival time of the hosts and in the generation time of the cells. When compared to the changes observed in the hosts of unirradiated leukaemic cells, the decrease in erythrocyte counts was greater during the first few days after inoculation of irradiated tumour cells, whereas the increase in leucocyte counts was slower.

The radiosensitivity of lymphosarcoma cells as determined by the liver colony method.

The liver colony method is based on the observation that intravenous injection of an appropriate number of LS/BL cells into isologous non-irradiated hosts leads to the formation of colonies of proliferating cells in the livers of these animals. The relationship between the number of cells injected and the number of colonies appearing in the livers was determined. This technique was used to measure the radiation sensitivity of LS/BL cells and it yielded a Do of 1.05 Gy. The results show that LS/BL cells have a similar radiation sensitivity as other mammalian cells. The liver colony assay used to determine the radiation sensitivity of the lymphosarcoma LS/BL cells can also be used whenever the number of viable tumour cells in a suspension is to be estimated.

A comparison of chromatin degradation in irradiated normal and tumorous lymphoid cells.

Irradiation of mice with doses of 2 and 4 Gy induced extensive chromatin degradation in the thymocytes within 6 hours accompanied by an increase in polydeoxynucleotide (PDN) content (36 and 42 times, respectively). Fifteen hours after irradiation the PDN level was considerably lower, however, still being 4.7 and 14 times the control values after doses of 2 and 4 Gy. The PDN content in control LS/BL lymphosarcoma cells was similar as that in the thymocytes of non-irradiated mice. Unlike in the thymocytes, irradiation of lymphosarcoma cells did induce no statistically significant increase in the PDN level 6 and 15 hours after the irradiation, respectively. It has been reported previously (Matyásová et al. 1973) that chromatin of LS/BL cells degraded similarly as that in the irradiated thymocytes. The results of the present experiments thus provide additional evidence for changes of LS/BL cell properties due to long term cultivation. These cells, however, are still able to react by chromatin fragmentation to nitrogen mustard treatment.

The effects of radiation and alkylating agents on chromatin degradation in normal and tumour lymphoid cells.

The dynamic of chromatin degradation was studied in thymocytes and LS/BL tumour cells. In permeabilised LS/BL cells, the rate of DNA degradation induced by endogenous calcium and magnesium-dependent endonuclease was approx. 25 times slower than in thymocytes. In LS/BL cells irradiation does not induce chromatin degradation. The alkylating agent TS 160 induced chromatin degradation in both LS/BL lymphosarcoma cells and thymocytes.