Three pairs of human tumour cell lines, with one line of each pair resistant to cisplatin, were used to compare the effects of cisplatin and ZD0473 on cellular toxicity and radiosensitization. Whilst all three cell line pairs had one line that was resistant to cisplatin, for ZD0473 the lung tumour HTB56 cp and cervical carcinoma ME180 cell lines did not express resistance to their HTB56 and SHA counterparts, respectively. Only the ovarian carcinoma line A2780 cp showed resistance to ZD0473 compared to its counterpart A2780 s . For radiosensitization both cisplatin and ZD0473 show additive and subadditive effects in the ovarian carcinoma lines, and additive and superadditive effects in the cervical carcinoma and lung tumour cell lines. In fact in the lung tumour cell lines ZD0473 appeared to be a more effective radiosensitizer than cisplatin.
Mouse embryo cells (C3H 10T1/2) were transfected with a plasmid (pAL8A) containing the HRas oncogene and neomycin resistance gene. Five transfected cell clones were isolated and established as cell lines, and these showed neomycin resistance. Two of these cell lines expressed a normal morphology while three showed a transformed morphology. Four of the cell lines produced tumours in nude mice. Flow cytometry measurements showed that exponentially growing cell cultures of the five transfected cell lines had the same cell cycle distribution as the normal parental cell line. The sensitivity to hyperthermia of the five transfected cell lines was the same as that of the normal cell line for temperatures ranging from 42.0 to 45.0°C. Thus in these studies we found no difference in the thermal sensitivity of normal and malignant cells transfected by the Hras oncogene.
The response of cultured CHO cells to U.V.L. irradiation during treatment with anisotonic solutions shows that treatment with hypotonic sucrose, NaCl or KCl solutions causes an increase in the cellular U.V.L. sensitivity, while exposure to hypertonic solutions causes a large decrease in U.V.L. sensitivity. Cells exposed to 1·8 M sucrose, NaCl or KCl solutions and given a U.V.L. dose of 252 erg/mm2 towards the end of the 20 min solution exposure time have survival levels which are respectively 228, 26, and 23 times higher than the controls, i.e. cells irradiated in phosphate buffered saline. Cell volume data obtained using a Coulter counter, and nuclear area data of attached cells obtained using an optical microscope with a micrometer reticle, show that cell and nuclear size are related to U.V.L. sensitivity. That is, as cells shrink and the nuclear area decreases, the cells become more U.V.L.-resistant. During hypotonic treatment with 0·1 M NaCl, the cell volume, nuclear area and U.V.L. sensitivity increased in the first 2 to 4 min of exposure time, but at longer exposure times (greater than 3 to 4 min), cell volume, nuclear area and cellular U.V.L. sensitivity decreased. For 0·1 M KCl treatment the cells initially displayed a rapid increase in volume, nuclear area and U.V.L. sensitivity, but at the longer exposure times no decrease in cell and nuclear size were observed, and a slight increase in U.V.L. sensitivity occurred. Changes in U.V.L. sensitivity were related to changes in nuclear size and cell volume; however, calculations showed that during hypertonic treatment there is an ionic effect as well as an osmotic effect. That is, the cellular U.V.L. survival in equal hypertonic concentrations of NaCl or KCl was lower than in the same concentration of sucrose.
SummaryThe response of cultured CHO cells to U.V.L. irradiation during treatment with anisotonic solutions shows that treatment with hypotonic sucrose, NaCl or KCl solutions causes an increase in the cellular U.V.L. sensitivity, while exposure to hypertonic solutions causes a large decrease in U.V.L. sensitivity. Cells exposed to 1·8 M sucrose, NaCl or KCl solutions and given a U.V.L. dose of 252 erg/mm2 towards the end of the 20 min solution exposure time have survival levels which are respectively 228, 26, and 23 times higher than the controls, i.e. cells irradiated in phosphate buffered saline.Cell volume data obtained using a Coulter counter, and nuclear area data of attached cells obtained using an optical microscope with a micrometer reticle, show that cell and nuclear size are related to U.V.L. sensitivity. That is, as cells shrink and the nuclear area decreases, the cells become more U.V.L.-resistant. During hypotonic treatment with 0·1 M NaCl, the cell volume, nuclear area and U.V.L. sensitivity increased in the first 2 to 4 min of exposure time, but at longer exposure times (greater than 3 to 4 min), cell volume, nuclear area and cellular U.V.L. sensitivity decreased. For 0·1 M KCl treatment the cells initially displayed a rapid increase in volume, nuclear area and U.V.L. sensitivity, but at the longer exposure times no decrease in cell and nuclear size were observed, and a slight increase in U.V.L. sensitivity occurred. Changes in U.V.L. sensitivity were related to changes in nuclear size and cell volume; however, calculations showed that during hypertonic treatment there is an ionic effect as well as an osmotic effect. That is, the cellular U.V.L. survival in equal hypertonic concentrations of NaCl or KCl was lower than in the same concentration of sucrose.
Hyperthermia has been shown in many studies to be a strong sensitizer for cisplatin treatment and this sensitization may be in part due to the inhibition of DNA repair processes. We have set out to test this in cells with specific gene knockouts for known repair processes. The chicken DT40 cell system was used with a parental line (DT40) and knockouts of homologous recombination (HR) repair DT40Rad54, nonhomologous recombination endjoining (NHEJ) repair (DT40Ku70) and a double knockout mutant DT40Ku70Rad54. The results show that thermal cisplatin sensitization was achieved in all cell lines when hyperthermia at 45°C for 1.5h was given before cisplatin treatment and 42°C hyperthermia was given concurrently with cisplatin treatment. The data show that inhibition of the HR repair system did not significantly affect sensitization, while inhibition of NHEJ reduced thermal sensitization at low cisplatin doses and short treatments and for concurrent treatments. These data indicate that there may be a partial involvement of NHEJ in thermal cisplatin sensitization under specific treatment conditions. Keywords: cisplatin, hyperthermia, homologous recombination, nhej
The effect of hyperthermia on the inhibition of potentially lethal radiation damage repair (PLDR) was measured in two human melanoma cell lines of differing radiosensitivities. The HT144 cell line is radiation sensitive and has an exponential survival curve, while the SK-MEL-3 line is radiation resistant and has a shouldered survival curve. Both cell lines demonstrated a similar level of PLDR at isosurvival level radiation doses of 2.0 and 10 Gy for the HT144 and SK-MEL-3 lines, respectively. Hyperthermia (40-45 degrees C) could inhibit PLDR in both cell lines. This inhibition was greater in HT144 than in SK-MEL-3 at low temperatures (40 degrees C) and was reversed at higher temperatures (45 degrees C). There was also a sequence dependence of PLDR inhibition which showed a greater inhibition for heat before irradiation at 40 degrees C but the reverse for temperatures of 41 degrees, 43 degrees and 45 degrees C. These data show that hyperthermia can be an effective inhibitor of PLDR in both radiosensitive and resistant cell lines and that the degree of inhibition is not related to radiosensitivity.
The feasibility of chemically-induced premature chromosome condensation (PCC) was tested in two human fibroblast and two human malignant melanoma cell lines. To induce PCCs, calyculin A, a potent protein phosphatase inhibitor, was used at a final concentration of 80 nM. Attached cells and cells put into suspension by trypsinization were incubated with calyculin A at 37 degrees C for 1 hour. Calyculin A was able to induce PCCs in all the phases of the cell cycle with the tumour cell lines giving the highest PCC frequency. No systematic differences were observed between attached cells and cells put into suspension by trypsinization. However, a cytotoxic effect that led to the loss of 50% to 80% of the treated cells was observed. The cytotoxic effect was more severe in the fibroblast than in the tumour cell lines. The appearance of deformed, fragile and fragmented nuclei with no particular chromatin condensation would explain to some extent this cytotoxic effect. A better understanding of the mechanism of action of calyculin A would help generalizing its use to study interphase chromosome aberrations.
