Death-resistant and nonresistant malignant human cell lines under anoxia in vitro.

BACKGROUND: Erythropoietin supports the survival of erythroblasts. We previously demonstrated that 24 malignant human cell lines expressed erythropoietin and its receptor and that erythropoietin secretion was enhanced under anoxia. In this study, we examined the viability of 22 of these cell lines excluding two leukemia cell lines under anoxia. METHODS: Twenty-two cancer cell lines of various origins were cultured under anoxia or normoxia for 4 days, and their viability was examined at 1-day intervals. The levels of lactate and ATP were measured. The expressions of hypoxia-inducible transcription factor 1alpha (HIF-1alpha) and Bcl-2 family proteins were examined by western blotting analysis. The cellular and mitochondrial features were examined by microscopy. RESULTS: Eleven of the 22 cancer cell lines examined showed 80% to 100% cell viability after 4 days under anoxia; 2 cell lines showed similar viability for 3 days, 3 cell lines showed similar viability for 2 days, and 6 cell lines showed similar viability for 1 day or less. These 11 death-resistant cell lines, which secrete various amounts of erythropoietin under anoxia, produced significantly more lactate during 2 days under anoxia than under normoxia, with ATP levels about 60% of those before anoxia. ATP returned to the normal level when normoxia was restored after 4 days of anoxia. However, the nonresistant cell lines responded to anoxia by yielding significantly more lactate without a reduction of the ATP level. The expression patterns of Bcl-2 family proteins revealed that apoptosis-inhibiting signals predominated over proapoptotic signals in the death-resistant cells under anoxia. CONCLUSION: The majority of the cancer cell lines examined survived under anoxia in vitro, through the Pasteur effect, in a dormant state without direct support of erythropoietin.

Split dose recovery studies using homologous recombination deficient gene knockout chicken B lymphocyte cells.

To understand the role of proteins involved in DSB repair modulating SLD recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52 (-/-) cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C(-/-) cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54(-/-) cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52(-/-), XRCC2(-/-), XRCC3(-/-) RAD51C(-/-) and RAD51D(-/-)) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52(-/-) was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2 (-/-), XRCC3(-/-), RAD51C (-/-) and RAD51D(-/-) knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D(-/-) and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2(-/-) , XRCC3(-/-), RAD51C(-/-) and RAD51D(-/-) and further delayed when compared with that of RAD52(-/-) and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery.

DNA-PK: the major target for wortmannin-mediated radiosensitization by the inhibition of DSB repair via NHEJ pathway.

The effect of wortmannin posttreatment was studied in cells derived from different species (hamster, mouse, chicken, and human) with normal and defective DNA-dependent protein kinase (DNA-PK) activity, cells with and without the ataxia telangiectasia (ATM) gene, and cells lacking other regulatory proteins involved in the DNA double-strand break (DSB) repair pathways. Clonogenic assays were used to obtain all results. Wortmannin radiosensitization was observed in Chinese hamster cells (V79-B310H , CHO-K1), mouse mammary carcinoma cells (SR-1), transformed human fibroblast (N2KYSV), chicken B lymphocyte wild-type cells (DT40), and chicken Rad54 knockout cells (Rad54-/-). However, mouse mammary carcinoma cells (SX9) with defects in the DNA-PK and chicken DNA-PK catalytic subunit (DNA-PKcs) knockout cells (DNA-PKcs-/-/-) failed to exhibit wortmannin radiosensitization. On the other hand, SCID mouse cells (SC3VA2) exposed to wortmannin exhibited significant increases in radiosensitivity, possibly because of some residual function of DNA-PKcs. Moreover, the transformed human cells derived from AT patients (AT2KYSV) and chicken ATM knockout cells (ATM-/-) showed pronounced wortmannin radiosensitization. These studies demonstrate confirm that the mechanism underlying wortmannin radiosensitization is the inhibition of DNA-PK, but not of ATM, thereby resulting in the inhibition of DSB repair via nonhomologous endjoining (NHEJ).

Erythropoietin regulates tumour growth of human malignancies.

In addition to the chief function of erythropoietin (Epo) in promoting erythropoiesis, some other roles have been found in the brain and uterus. We have reported that signalling pathways of Epo and Epo receptor (EpoR) are involved in the tumourigenesis of ovarian and uterine cancers. To determine whether Epo plays a similar role in other malignancies, we studied the expression of Epo in several malignant human cell lines. We found that 24 malignant human cell lines examined express Epo and EpoR regardless of their origins, types, genetic characteristics and biological properties and secrete a very small amount of Epo individually and that most of them respond to hypoxic stimuli by enhanced secretion of Epo. To determine whether the Epo-EpoR pathway operates in tumours of these cell lines, we transplanted several cell lines into nude mice and confirmed the presence of Epo-responsive sites in xenografts in which the phosphorylation of the STAT5 (signal transducer and activator of transcription) is detectable. Furthermore, in nude mice we blocked the Epo signalling in xenografts of two representative cell lines, stomach choriocarcinoma and melanoma, by i.p. injections of EpoR antagonist and found inhibition of angiogenesis and survival of tumour cells leading to destruction of tumour masses and disturbances of phosphorylation of STAT5. In contrast, Epo mimetic peptide promotes angiogenesis and tumour cell survival. These findings suggest that Epo is indispensable for the growth and viability of malignant tumour and also that the deprivation of Epo signalling may be a promising therapy for human malignancy.