Reduced expression of the ICE-related protease CPP32 is associated with radiation-induced cisplatin resistance in HeLa cells.

H Eichholtz-Wirth,K Marx,O Stoetzer

doi:10.1038/bjc.1997.555

Abstract

Low-dose fractionated gamma-irradiation (three cycles of 5 x 2 Gy) induced cisplatin resistance in HeLa cells. The drug resistance was modest (Rf of about 2) and stable, similar to that found previously in murine cells after irradiation. In the drug-resistant HeLa-C3 cells, flow cytometric analysis revealed a decreased number of apoptotic cells compared with the parental cells. Drug resistance was associated with considerably enhanced expression of the p53 suppressor protein in HeLa-C3 cells after cisplatin exposure but seemed not to be regulated by the bcl-2-dependent pathway. Cisplatin resistance correlated with reduced expression of ICE-related proteases (interleukin-1beta-converting enzyme). Basal levels of the 45-kDa precursor ICE protein were reduced in HeLa-C3 cells, while those of the mature 60-kDa heterotetramer were similar. The CPP32 protease, a member of the ICE family with structural homology but different substrate specificity, was expressed at a lowered level. After drug exposure, there was a slight increase of CPP32 in HeLa-C3 cells, equivalent to about 45% of the level attained in the parental cells. This is in contrast to the CPP32 levels measured after irradiation, which were similar in sensitive and in resistant cells. As the radiosensitivity is unchanged in both cell lines, these results suggest that cisplatin resistance in HeLa-C3 cells is associated with alterations of a CPP32-linked apoptotic pathway, which is affected by the damage caused by cisplatin but not by irradiation. Whether these changes are dependent on the observed p53 modifications is now being studied in resistant clones.

Highlights

We have recently shown that cisplatin resistance is induced in murine fibrosarcoma cells by low-dose fractionated y-irradiation
For the induction of cisplatin resistance, exponentially growing HeLa cells were subjected to a cycle of fractionated 137CS irradiation (5 x 2 Gy in 7 days)
HeLa-C3 cells slightly changed their phenotype with more polyhedric morphology compared with the more elongated shape of the parental cells

Summary

Methods

MaterialsThe following drugs and chemicals were used: cisplatin solution (Medac, Hamburg); vincristine (Lilly), doxorubicin (Farmitalia), etoposide (Bristol), Eagle's minimal essential medium (Serva); bycomycin (Byc Gulden, Konstanz); newborn calf serum (CCPro, Karlsruhe); all other chemicals were purchased from Sigma Chemie, Deisenhofen.For the Western blot analysis, the following antibodies were used: precICE and ICE, plO(C-20) rabbit PAb (Santa Cruz); p53 DO-7 and bcl-2, mouse MAb (Dako); CPP32, mouse MAb (Transduction Laboratories). The following drugs and chemicals were used: cisplatin solution (Medac, Hamburg); vincristine (Lilly), doxorubicin (Farmitalia), etoposide (Bristol), Eagle's minimal essential medium (Serva); bycomycin (Byc Gulden, Konstanz); newborn calf serum (CCPro, Karlsruhe); all other chemicals were purchased from Sigma Chemie, Deisenhofen. HeLa and HeLa-C3 cells were grown as monolayer cultures in Eagle's minimal medium, containing L-glutamine and supplemented with 10% newborn calf serum, 0.01% bycomycin and 0.035% sodium bicarbonate and maintained at 37°C at pH 7.4 in a controlled atmosphere (3-3.5% carbon dioxide in air). The cells were subcultured once per week over 4-6 weeks with parallel checking of drug sensitivity. If the drug sensitivity was unchanged, the treatment protocol was repeated. The preirradiated cells were denoted Cl, C2, C3, according to the number of treatment cycles

Results

Discussion

Conclusion