Response of base excision repair enzymes to complex DNA lesions.

There is now increasing evidence that ionizing radiation generates complex DNA damage, i.e. two or more lesions--single-strand breaks or modified nucleosides--located within one to two helical turns on the same strand or on opposite strands. Double-strand breaks are the most readily recognizable clustered lesions, but they may constitute a relatively minor fraction of the total. It is anticipated that clustered lesions may play a significant role in cellular response to ionizing radiation since they may present a major challenge to the DNA repair machinery. The degree of lesion complexity increases with increasing LET. This has potential implications for space travel because of exposure to high-LET cosmic radiation. It is therefore critical that we begin to understand the consequences of such damaged sites, including their influence on DNA repair enzymes. This paper presents a short review of our current knowledge of the action of purified DNA repair enzymes belonging to the base excision repair pathway, including DNA glycosylases and apurinic/apyrimidinic endonucleases, on model complex lesions.

Biological factors influencing the RBE of neutrons: implications for their past, present and future use in radiotherapy.

The recent resurgence of interest in fast-neutron therapy, particularly for the treatment of prostate cancer, warrants a review of the original radiobiological basis for this modality and the evolution of these concepts that resulted from subsequent experimentation with the fast-neutron beams used for randomized clinical trials. It is clear from current radiobiological knowledge that some of the postulates that formed the mechanistic basis for past clinical trials were incorrect. Such discrepancies, along with the inherent physical disadvantages of neutron beams in terms of collimation and intensity modulation, may partially account for the lack of therapeutic benefit observed in many randomized clinical trials. Moreover, it is equally apparent that indiscriminate prescription of fast-neutron therapy is likely to lead to an adverse clinical outcome in a proportion of patients. Hence any renewed efforts to establish a niche for this modality in clinical radiation oncology will necessitate the development of a triage system that can discriminate those patients who might benefit from fast-neutron therapy from those who might be harmed by it. In the future, fast-neutron therapy might be prescribed based upon the relative status of appropriate molecular parameters that have a differential impact upon radiosensitivity to photons compared to fast neutrons.

Differential impact of Raf-1 kinase activity on tumor cell resistance to paclitaxel and docetaxel.

Docetaxel (Taxotere) is becoming increasingly important in the treatment of many tumor sites and is unusually active in tumors that are resistant to the structurally similar taxane, paclitaxel. These data suggest that the processes that confer cellular paclitaxel resistance may have a substantially lower impact upon the cytotoxicity induced by docetaxel. We have recently reported that there is a marked Raf-1 kinase dependency of paclitaxel resistance in human cervical and ovarian tumor cell lines. We therefore characterized the impact that inherent and genetically induced variations in Raf-1 kinase activity have on the docetaxel cytotoxicity in human ovarian and cervical cancer cell lines. Our data suggest that docetaxel cytotoxicity is independent of Raf-1 kinase activity in the cell lines studied and that the lack of cross-resistance between these two taxane compounds may be due to the differential impact that Raf-1 kinase activity has on their cytotoxicity. Should these relationships pertain to the clinical situation, these findings could form the basis for a molecular-based triage of patients to receive docetaxel when response to paclitaxel may be unlikely due to high Raf-1 kinase activity.

ERCC1 expression as a molecular marker of cisplatin resistance in human cervical tumor cells.

Cisplatin is a valuable adjuvant to radiotherapy for the treatment of cervical cancer. Because the advantage of combining cisplatin with radiotherapy is likely to be attributable to additive cell killing by these 2 agents, such protocols should primarily benefit patients who have inherently cisplatin-sensitive tumors. Development of a molecular assay to rapidly evaluate the cisplatin responsiveness of cervical tumors would thus be extremely valuable. We investigated whether high pre-treatment mRNA levels of the ERCC1 nucleotide excision repair gene are predictive of cisplatin resistance in early-passage human cervical cancer cells, as they are in several other tumor types. Expression of the ERCC1 gene at the mRNA and protein levels was established by Northern and Western blotting, respectively, in a panel of single-cell-derived cervical carcinoma cell lines that exhibited a wide range of inherent sensitivity to cisplatin. There was a significant (p

The influence of hypoxia on the relative sensitivity of human tumor cells to 62.5 MeV (p-->Be) fast neutrons and 4 MeV photons.

Fast neutrons have been used in the clinical radiation therapy of tumors largely because of experimental evidence that their cytotoxic effects are much less dependent on oxygen levels than those of low-LET photons. The potential therapeutic advantage of fast neutrons based on hypoxia alone can be calculated as the "hypoxic gain factor", which is the ratio of the OERs for the fast-neutron compared to the photon beams. The hypoxic gain factor that is generally anticipated based on studies with established mammalian cell lines is about 1.6. However, surprisingly few studies have examined the influence of hypoxia on the fast-neutron radiosensitivity of human tumor cells of different histological types. For this reason, we have determined the OERs of five human tumor cell lines exposed to 62.5 MeV (p-->Be) cyclotron-generated fast neutrons or 4 MeV photons from a clinical linear accelerator. The OERs for four chemotherapy-naive cell lines, HT29/5, Hep2, HeLa and RT112, were invariably greater for photons than for neutrons, but all of these values were lower than expected on the basis of the previous literature. Despite their low OERs, these cell lines showed hypoxic gain factors that were within the range of 1.31-1.63, indicating that such effects cannot entirely explain the disappointing clinical results obtained with fast neutrons. In contrast, comparison of the surviving fractions at clinically relevant doses (1.6 Gy of neutrons and 2.0 Gy of photons) for these four tumor cell lines suggested that little benefit should result from neutron treatment. Only the cisplatin-resistant OAW42-CP line showed a significant hypoxic gain factor by this method of analysis. We conclude that, at the dose fractions used in clinical radiation therapy, there may not be a radiobiological precedent for higher local control rates after fast-neutron irradiation of hypoxic tumor cells.

Raf-1 kinase activity predicts for paclitaxel resistance in TP53mut, but not TP53wt human ovarian cancer cells.

We have recently reported that there is a significant Raf-1 kinase dependency of paclitaxel resistance in human cervical tumor cell lines. In light of the possibility that Raf-1 kinase inhibitors could be used to enhance paclitaxel responsiveness in ovarian cancer, we have characterized the Raf-1 kinase dependency of paclitaxel resistance in ovarian cancer cells. The relationship between Raf-1 kinase activity and the sensitivity to clinically relevant paclitaxel concentrations was determined in four ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/WT and OAW42/WT). Furthermore, in recognition that such a drug combination would initially be used in patients whose tumors have recurred following cisplatin/paclitaxel treatment, we also determined the Raf-1 kinase dependency of paclitaxel cytotoxicity in cisplatin resistant variants of two of the ovarian cell lines (2780/CP and OAW42/CP). In the two cell lines (2780/WT and OAW42/WT) that possess a wild-type TP53 (TP53wt), the relationship between Raf-1 kinase activity and paclitaxel resistance was different from that observed in the cervical tumor cell lines. In these cell lines, paclitaxel-induced far more cell killing than would have been predicted from their Raf-1 kinase activity. However, in the ovarian cancer cell lines (CA-OV3, SK-OV3, 2780/CP and OAW42/CP) that have a mutant TP53 (TP53mut), the cytotoxicity induced by 60 nM paclitaxel exhibited the same relationship to Raf-1 kinase activity as previously observed in cervical tumor cell lines. These data suggest that the therapeutic efficacy of paclitaxel in ovarian cancer patient whose tumors have TP53mut might be increased if it is administered in combination with Raf-1 kinase inhibitors, e.g., ISIS 5132.

DNA double-strand break rejoining in human follicular lymphoma and glioblastoma tumor cells.

Follicle center cell lymphoma is among the most radioresponsive of human cancers. To assess whether this radioresponsiveness might be a result of a compromised ability of the tumor cells to accomplish the biologically-effective repair of DNA double-strand breaks (DSBs), we have measured i) the extent of the mechanical rejoining of radiation-induced DSBs in biopsy-derived follicle center cell lymphoma cells and ii) the fidelity with which nuclear protein extracts from these cells rejoin restriction enzyme-induced DSBs. Cell suspensions derived from two lymphoma biopsies, designated FCL1 and FCL2, as well as two established human glioblastoma cell lines, M059J and M059K, were exposed to 30 Gy of gamma-rays and evaluated for their ability to rejoin DSBs using a Southern transfer-pulsed-field gel electrophoresis assay. The fidelity of rejoining of restriction enzyme-induced DSBs was assessed using a cell-free plasmid reactivation assay. Both lymphoma suspensions rejoined DSBs relatively slowly and exhibited a similar phenotype to the known DSB-rejoining deficient M059J line. The level of DSB mis-rejoining in the cell-free plasmid reactivation assay was also similar in M059J and FCL2 cells and was considerably ( approximately 6-fold) higher than in M059K cells. Because of insufficient numbers of cells, we were unable to perform this assay with the FCL1 lymphoma. These limited data suggest that follicle center cell lymphoma cells may be intrinsically deficient in performing the biologically-effective rejoining of DSBs. Such a deficiency might contribute to the radioresponsiveness of this disease and may be exploitable in the development of improved treatment strategies, such as radioimmunotherapy.

Modification of non-conservative double-strand break (DSB) rejoining activity after the induction of cisplatin resistance in human tumour cells.

The induction of collateral radioresistance after the development of cisplatin resistance is a well-documented phenomenon; however, the exact processes that are responsible for the cisplatin-induced radioresistance remain to be elucidated. There was no obvious difference in the level of radiation-induced DNA double strand breaks (DSBs), in DSB rejoining rates, or the level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in the cisplatin- and radiation-sensitive 2780/WT and cisplatin-resistant 2780/CP cell lines. However, there was a significantly (P < 0.01) lower level of DSB misrejoining activity within nuclear protein extracts derived from the cisplatin- and radiation-sensitive 2780/WT and OAW42/WT tumour cell lines than in similar extracts from their cisplatin- (and radiation-) resistant 2780/CP and OAW42/CP counterparts. All of the DSB misrejoining events involved deletions of between 134 and 444 bp that arose through illegitimate recombination at short repetitive sequences, such as those that arise through non-homologous repair (NHR). These data further support the notion that the radiosensitivity of DSB repair proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways. Furthermore, our data suggest that the induction of acquired cisplatin resistance is associated with a two- to threefold decrease in the activity of a non-conservative DSB rejoining mechanism that appears to be a manifestation of NHR.

High Raf-1 kinase activity protects human tumor cells against paclitaxel-induced cytotoxicity.

Paclitaxel (Taxol) is becoming increasingly important in the treatment of many tumors, although a large proportion of tumors fail to respond to this drug. The identification of the processes that confer cellular paclitaxel resistance could provide potential targets for novel therapies that may help to eliminate paclitaxel-resistant tumors. Recent reports suggest that the Raf-1 protein kinase may have a profound influence on the level of paclitaxel-induced apoptosis. We have critically evaluated the relationship between Raf-1 kinase activity and de novo paclitaxel resistance in early-passage human cervical tumors. In the 12 cell lines studied, Raf-1 kinase activity was inversely correlated (P = 0.0016) with the level of cytotoxicity induced by 60 nM paclitaxel. The relationship between these two parameters seems to be more than an epiphenomenon, because genetic down-regulation of Raf-1 kinase activity led to an approximately 4-fold increase in paclitaxel-induced cytotoxicity. The data from both our transfection studies and those on the 12 unperturbed cell lines are consistent with Raf-1 kinase being a negative determinant of paclitaxel-induced cytotoxicity. Because the cytotoxicity of paclitaxel is primarily attributable to apoptosis, these data suggest that Raf-1 kinase acts to suppress paclitaxel-induced apoptosis. These data suggest that the clinical effectiveness of paclitaxel could be substantially improved by the use of Raf-1 kinase inhibitors, provided that a similar relationship between Raf-1 kinase activity and paclitaxel cytotoxicity exists in the clinic, especially in those tumor sites where paclitaxel is the current treatment of choice e.g., ovarian and breast cancer.

Paclitaxel is preferentially cytotoxic to human cervical tumor cells with low Raf-1 kinase activity: implications for paclitaxel-based chemoradiation regimens.

BACKGROUND AND PURPOSE: Low Raf- kinase activity has been reported to be associated with radioresistance in epithelial tumor cell lines and with paclitaxel sensitivity in cervical tumor cells. Paclitaxel might thus be effective in eliminating radioresistant clones from cervical tumors, even in the absence of synergistic interaction between these therapeutic modalities. We thus established the relationship between Raf-1 kinase activity and radiosensitivity in human cervical tumor cells and determined if paclitaxel is preferentially cytotoxic to radio-resistant tumor clones. MATERIALS AND METHODS: We established and contrasted the radiation and paclitaxel sensitivity of 12 human cervical tumor clones that exhibited a wide range of Raf-1 kinase activity. RESULTS: Raf-1 kinase activity was inversely correlated (P = 0.001) with SF2 values in the 12 cervical tumor clones studied. Paclitaxel was preferentially cytotoxic to radioresistant tumor clones, with the level of paclitaxel-induced cytotoxicity being significantly (P = 0.0016) influenced by Raf-1 kinase activity levels. CONCLUSIONS: Our in vitro data indicate that there are marked, but completely opposite, Raf-1 kinase dependencies of radiation and paclitaxel cytotoxicity in cervical tumor cells. The use of combined paclitaxel and radiotherapy treatment may thus lead to higher local control rates for squamous cell carcinoma of the cervix. Circumstantially, our data suggest that the greatest therapeutic gains might accrue if paclitaxel was administered when there is the greatest proportion of tumor clones with low Raf-1 kinase activity. It may thus be desirable to use paclitaxel towards the end of radiotherapy treatment or post-radiotherapy as consolidation therapy.

Constancy of the relative biological effectiveness of 42 MeV (p-->Be+) neutrons among cell lines with different DNA repair proficiencies.

An important approach to understanding the role of the various DNA repair pathways in the cellular response to DNA-damaging agents is through the study of repair-deficient mutant cell lines. In the present study we used this strategy to assess the relative importance of four of these pathways for the repair of DNA damage induced by low-linear energy transfer (LET) gamma rays and intermediate-LET 42 MeV (p-->Be+) fast neutrons. The panel of hamster cell mutants that we characterized for their relative sensitivity to fast neutrons and gamma rays includes cell lines with defects in the nucleotide excision repair pathway; these can be further subdivided into mutants which are defective in nucleotide excision repair alone [UV5 (ERCC2-), UV24 (ERCC3-), UV135 (ERCC5-) and UV61 (ERCC6-)] compared to those which have an associated defect in the distinct but overlapping pathway for the repair of DNA crosslinks [UV20 (ERCC1-) and UV41 (ERCC4-)]. We also examined mutants with defects in the base excision repair pathway [EM9 (XRCC1-)] and the DNA-dependent protein kinase (DNA-PK)-mediated DNA double-strand break (DSB) repair pathway [xrs5 (XRCC5-)]. None of the mutants defective in nucleotide excision repair was differentially sensitized to fast neutrons or gamma rays; in fact, the slight radiosensitivity of these mutants under aerated conditions may be secondary to their defect in nucleotide excision repair. In contrast, deficiency in the base excision repair pathway resulted in a significant primary sensitization to both types of radiation (1.95-fold to gamma rays and 1.79-fold to neutrons). Deficiency in the DSB repair pathway mediated by DNA-PK resulted in a marked, but again similar, primary sensitization to gamma rays (4.2-fold) and neutrons (5.1-fold). Thus none of the repair pathways examined here exhibited a preferential role for the repair of damage induced by low-LET compared to intermediate-LET radiations; this resulted in an essentially constant relative biological effectiveness (RBE) of approximately 2 among the cell lines studied, independent of their DNA repair proficiency. However, consideration of these data along with data published previously for high-LET alpha particles suggests that, whereas the DNA-PK pathway is important for the repair of DSBs induced by low- and intermediate-LET radiations, it becomes less important as the LET increases beyond 100 keV/microm; thus this pathway may not be involved in repairing the more complex lesions induced by densely ionizing high-LET particles.

Differential level of DSB repair fidelity effected by nuclear protein extracts derived from radiosensitive and radioresistant human tumour cells.

A cell-free plasmid reactivation assay was used to determine the fidelity of DNA double-strand break (DSB) repair in a panel of eight DSB repair-proficient human tumour cell lines. Nuclear protein extracts derived from radiosensitive tumour cells were less capable of correctly rejoining EcoRI-induced DSBs than were similar extracts from radioresistant tumour cells. Linear regression analysis suggests that there was a significant (r2 = 0.84, P = 0.001, d.f. = 6) correlation between the fidelity of DSB rejoining and the SF2 values of the cell lines studied. This cell-free assay is clearly sensitive to differences in the nuclear protein composition that reflect the clinically relevant radiosensitivity of these cell lines. The fact that our cell-free assay yielded similar results to previous studies that used intracellular plasmid reactivation assays suggests that those differences in DSB mis-rejoining frequencies in radiosensitive and radioresistant cell lines may be due to inherent differences in nuclear protein composition and are not directly attributable to differences in proliferation rates between cell lines. The underlying cause for this association between DSB mis-rejoining frequencies and radiosensitivity is presently unknown, however restriction endonuclease mapping and polymerase chain reaction (PCR) amplification analysis revealed that approximately 40% of the mis-rejoined DSBs arose as a result of the deletion of between 40 and 440 base pairs. These data raise the possibility that the radiosensitivity of DSB repair-proficient human tumour cell lines may be partly determined by the predisposition of these cell lines to activate non-conservative DSB rejoining pathways.

Intratumoral heterogeneity as a confounding factor in clonogenic assays for tumour radioresponsiveness.

The level of intra-tumoral heterogeneity of cellular radiosensitivity within primary cultures of three carcinomas of the cervix has been established. All three cultures contained clones that varied by as much as 3-fold in their clinically relevant radiosensitivity (SF2). The level of intra-tumoral heterogeneity observed in these cervical tumour cultures was sufficient to be a major confounding factor to the use of pre-treatment assessments of radiosensitivity to predict for clinical radioresponsiveness. Mathematical modeling of the relative elimination of the tumour clones during fractionated radiotherapy indicates that, in two of the three biopsy samples, the use of pre-treatment derived SF2 values from the heterogeneous tumour sample would significantly overestimate radioresponsiveness. We conclude that assays of cellular radiosensitivity that identify the radiosensitivity of the most radioresistant clones and measure their relative abundance could potentially increase the effectiveness of SF2 values as a predictive marker of radioresponsiveness.

Effect of cisplatin on the clinically relevant radiosensitivity of human cervical carcinoma cell lines.

PURPOSE: To evaluate the effect of clinically relevant levels of cisplatin on the radiosensitivity of human cervical tumor cells, and to estimate what changes in local control rates might be expected to accrue from the concomitant use of cisplatin during fractionated radiotherapy. METHODS AND MATERIALS: The effects of concomitant cisplatin (1 microgram/ml, a typical intratumor concentration) on the clinically relevant radiosensitivity, i.e., surviving fraction after 2 G (SF2) values, was determined in 19 cloned human cervical tumor cell lines. These early passage cell lines had SF2 values ranging from 0.26 to 0.87. RESULTS: The concomitant administration of cisplatin reduced the clinically relevant radiosensitivity in the majority (11 out of 19) of the human tumor cell lines investigated. In only 4 out of 19 was any radiosensitization observed, and in 4 out of 19 cell lines there was no significant change in radiosensitivity. However, the sum of the independent cell killing by radiation and cisplatin, was approximately twofold higher than after radiation alone. There was no apparent dependence of the cisplatin-induced changes in SF2 values upon the level of cell killing by cisplatin. However, there is a suggestion that concomitant cisplatin administration may have a differential effect in inherently radiosensitive and resistant human tumor cell lines. CONCLUSIONS: Our data suggest that concomitant cisplatin/radiotherapy regimens may result in a higher level of local tumor control, but primarily through additive toxicity and not through radiosensitization. Future improvements in local tumor control may, thus, be derived by increasing the total dose of cisplatin.

Mechanisms of cyclophosphamide resistance in a human myeloid leukemia cell line.

The 4-hydroperoxycyclophosphamide (4HC)-resistant B5-180(3) subline of the cloned KBM-7/B5 cell line was developed as a model of induced cyclophosphamide resistance in human myeloid leukemia. Based on IC90 values, this subline was approximately 20-fold resistant to 4HC. Furthermore, it was significantly cross-resistant to phosphorodiamidic mustard (PM), whose cytotoxicity is independent of aldehyde dehydrogenase (ADH). Using alkaline elution we found that the resistant line had decreased initial levels of DNA interstrand cross-links (ISCs) following 4HC but not PM treatment. The resistant cells also appeared to remove ISCs from their DNA more rapidly than the parental cells. Our data therefore suggest that 4HC resistance in the B5-180(3) subline is multifactorial; ADH is an important mediator of its resistance to ISC induction by 4HC, while a second process, which may involve an increased ability to tolerate drug-induced DNA damage, appears to be important for its resistance to both 4HC and PM. The B5-180(3) cells were also cross-resistant to gamma-radiation (approximately 1.7-fold at a surviving fraction of 0.1); if generally applicable, such effects could have important clinical implications, since pretransplant total body irradiation is a major component of the eradication of leukemic cells.

The role of DNA damage in the resistance of human chronic myeloid leukemia cells to cyclophosphamide analogues.

We examined 4-hydroperoxycyclophosphamide (4HC) and phosphorodiamidic mustard (PM)-mediated cytotoxicity and DNA interstrand cross-link (ISC) induction in a 4HC-resistant subline, B5-180(3), that was derived from the subcloned human myeloid leukemia cell line KBM-7/B5. Based on the ratio of the 50% inhibitory concentration values, B5-180(3) cells were approximately 35-fold resistant to 4HC compared with the parental cells. 4HC-induced ISC levels (as measured by alkaline elution) were approximately 9-fold lower in the 4HC-resistant line than the parent line. To determine whether the approximately 9-fold reduction of initial 4HC-induced ISCs was related to elevated aldehyde dehydrogenase (ADH), we examined PM-mediated cytotoxicity and ISC induction in the two cell lines. Unlike 4HC, the cytotoxicity of PM is independent of ADH activity. B5-180(3) cells were between 5- and 7-fold resistant to the cytotoxic effects of PM and were only slightly (approximately 1.3-fold) resistant to PM-mediated ISC induction. Furthermore, when cells were exposed to 4HC in the presence of 40 micrograms/ml cyanamide, an efficient inhibitor of ADH, the resistance index based on the 50% inhibitory concentration values was decreased to approximately 8. Elevated ADH can therefore explain most of the decrease in 4HC-induced ISCs, with elevated GSH levels probably accounting for the remaining small increment of resistance. Thus, decreased levels of ISC induction can account for a factor of approximately 9 of the total approximately 35-fold resistance to 4HC. The remaining increment of resistance to 4HC, as well as the cross-resistance to PM, appears to relate to an enhanced ability of the resistant subline to tolerate drug-induced ISCs. Thus, multiple factors appear to be involved in the resistance of these cells to the cytotoxic and DNA-damaging effects of 4HC, with elevated ADH and temporal factors (related to the processing of DNA damage) being the most important of these.

Identification of human in vitro cell lines with greater intrinsic cellular radiosensitivity to 62.5 MeV (p-->Be+) neutrons than 4 MeV photons.

PURPOSE: To identify human in vitro cell lines with a high relative cellular sensitivity to fast neutrons as compared to photons and to examine their relationship to intrinsic photon radiosensitivity and cellular proliferation kinetics. METHODS AND MATERIALS: The clonogenic cell survival following exposure to low LET, 4 MeV photons or, high LET, 62.5 MeV (p-->Be+) fast neutrons and the cell kinetic parameters of 30 human in vitro cell lines, covering a wide range of histologies, were analyzed alone and with previously published data of Fertil and Malaise. The relative survival at 1.6 Gy of neutrons (SF1.6) compared to 2 Gy of photons (SF2) (the doses per fractions used in the Clatterbridge fast neutron studies) and the cell kinetic parameters of the 30 cell lines were also compared. The relative lethality of 62.5 MeV fast neutrons was assessed by comparing the ratio alpha neutrons/alpha photons to alpha photons or SF1.6 neutrons/SF2 photons to SF2 photons. Cellular proliferation kinetics were measured by flow cytometry following BrdU incorporation and the relationship of cellular proliferation to relative neutron lethality was measured by comparing the alpha neutron/alpha photon ratio to the labelling index (LI), potential doubling (Tpot) and ploidy. RESULTS: The majority of cell survival curves obtained following exposure to 62.5 MeV fast neutrons were curvilinear with beta values of similar order to those obtained with low LET 4 MeV photons. Comparison of alpha values for neutrons and photons revealed a relatively neutron sensitive subset of 9 out of 30 in vitro cell lines. This subset was not, however, distinguishable when 1.6 Gy of neutrons was compared to 2 Gy of photons. There was no correlation between cell survival with neutrons or photons and the cell kinetic parameters Tpot or LI or with DNA ploidy. CONCLUSIONS: The use of in vitro assays of neutron and photon radiosensitivity irrespective of cell kinetic parameters allows identification of neutron sensitive cell populations when the ratio of the alpha values for neutrons and photons is compared to the reciprocal of the alpha photon value. This relationship is not apparent when fractions of 2 Gy of photons are compared to 1.6 Gy of neutrons. Whether or not this identification can be borne out in fractionated regimes in the clinic remains to be proved.

In vitro studies of intrinsic cellular radiosensitivity following 4 MeV photons or 62.5 MeV (p-->Be+) neutrons. Potential implications for high LET therapy.

Recent studies of the intrinsic cellular sensitivity of 30 human in vitro cell lines to 4 MeV photons and 62.5 MeV (p-->Be+) neutrons have identified relatively neutron sensitive cell lines with high alpha values within the more resistant end of the photon radiation response range. Here we present data comparing the surviving fraction at 2 Gy of photons (SF2) to the surviving fractions at 1.6, 0.85 and 0.6 Gy of neutrons respectively (SF1.6 SF0.85 and SF0.6). With the ratio SF2/SF1.6 a negative trend can be seen between the probability of a preferential response to neutrons and relative photon resistance. With a ratio of SF2/SF0.6, however, a highly significant benefit for 62.5 MeV neutrons can be seen in the more photon resistant lines. We suggest further clinical studies to explore the potential relevance of these in vitro findings to the clinical situation should be undertaken.

The relative cellular radiosensitivity of 30 human in vitro cell lines of different histological type to high LET 62.5 MeV (p-->Be+) fast neutrons and 4 MeV photons.

It has been suggested that fast neutron therapy may have a role in the treatment of those tumours which lie within the most photon-resistant histological categories. A clinical radiobiological study by Battermann et al., however, did not support this hypothesis (Battermann, J.J. et al., Eur. J. Cancer 17: 539-548, 1981). Similarly, in a comparison of the intrinsic cellular radiosensitivity of 20 human in vitro cell lines with 4 MeV photons and 62.5 MeV (p-->Be+) neutrons, there was no correlation between RBE and photon sensitivity. However, because the range of histological cell types in this in vitro study did not include sufficient representatives of the most sensitive and resistant histological categories, it was not possible to examine the relationship between histology and the relative efficacy of fast neutrons compared with photons. The intrinsic radiosensitivity of a further 10 human in vitro cell lines has thus been measured and the results of all 30 cell lines used in a comparison of the relationship between relative neutron sensitivity and histology. These results together with those obtained by reanalysis of published data from a clinical study of the RBE of pulmonary metastases by Battermann et al. suggest that in the clinical situation, photon-resistant histology per se may not be a sufficient criterion for the choice of high LET irradiation and emphasize the need for predictive assays for individual tumours.

C-raf-1 proto-oncogene expression relates to radiosensitivity rather than radioresistance.

The transfection of several oncogenes, particularly c-raf-1, into mammalian in vitro cell lines has been reported to be associated with increased radioresistance. We have thus investigated (by scanning photodensitometry of western blots) the phenotypic expression of the c-raf-1, c-myc and c-ras protein products in 19 human in vitro cell lines, whose intrinsic cellular sensitivity to 4 MeV photon irradiation has also been determined. High levels of c-raf-1 proto-oncogene product expression did not correlate with increased cellular radioresistance, but rather showed a significant correlation with intrinsic cellular radiosensitivity to photon irradiation for alpha (r = 0.664, P = 0.002), and SF2 (r = -0.655, P = 0.002). There was no significant correlation for the ras family, c-myc or actin. These results conflict with those of previous studies in which transfection of the activated forms of the c-raf-1 oncogene were associated with increased radioresistance, and suggest the possibility that the full length proto-oncogene may influence cellular radiosensitivity in a different manner from that of the activated oncogene.