The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign

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.

We have previously reported a correlation between high endogenous expression of the protein product of the RAF-1 proto-oncogene, intrinsic cellular radiosensitivity and rapid exit from a G2/M delay induced by 2 Gy of gamma-irradiation. Raf1 is a positive serine/threonine kinase signal transduction factor that relays signals from the cell membrane to the MAP kinase system further downstream and is believed to be involved in an ionizing radiation signal transduction pathway modulating the G1/S checkpoint. We therefore extended our flow cytometric studies to investigate relationships between radiosensitivity, endogenous expression of the Raf1 protein and perturbation of cell cycle checkpoints, leading to alterations in the G1, S and G2/M populations after 2 Gy of gamma-irradiation. Differences in intrinsic radiosensitivity after modulation of the G1/S checkpoint have generally been understood to involve p53 function up to the present time. A role for dominant oncogenes in control of G1/S transit in radiation-treated cells has not been identified previously. Here, we show in 12 human in vitro cancer cell lines that late G1 accumulation after 2 Gy of radiation is related to both Raf1 expression (r = 0.91, P = 0.0001) and the radiosensitivity parameter SF2 (r = -0.71, P = 0.009).

Purpose:llre tumor suppressor gene p53 can mediate cell cycle arrest or apoptosis in response to DNA damage.Accumulating evidence suggests that it may also directly or indirectly lnthmnce the DNA mpalr machinery.In the present study, we investigated whether ~53, induced by DNA damage, could enhance the rejoining of double-strand DNA breaks.

We have previously noted that high endogenous expression of the protein product of the full-length RAF1 proto-oncogene is related to relative intrinsic cellular radiosensitivity in 19 human cells lines in vitro. This appeared to be unrelated to the parameters of cell kinetics. In rodent and human cell lines transfected with dominant oncogenes, including Myc and MYC, Hras and HRAS and SV40, increased radioresistance has been accompanied by increased delay in progress through the G2 phase of the cell cycle after irradiation. We have thus examined the putative relationship between RAF1 expression and postirradiation perturbation of G2 phase in six of the human cell lines for which data have been reported previously. These lines exhibit a wide range of both radiosensitivity and Raf1 protein levels as measured previously by Western blotting. We report here that the cell lines whose cells appear to exit more rapidly from G2 phase are more radiosensitive (r = 0.91, P = 0.01) and express high levels of Raf1 protein (r = -0.93, P = 0.006).

Although several oncogenes, including c-myc,ras and c-raf-1, have been implicated in cellular resistance to ionising radiation, there is less information relating oncogene expression to cis-diamminedochloroplatinum (CDDP) resistance. However, transfection of c-myc or v-H-ras and activation of protein kinase C (PKC), which contributes to the RAF-I, MAP kinase signal transduction pathway, can influence therapeutic response to CDDP. Activation of PKC increases CDDP sensitivity, whilst transfected c-myc or v-H-ras induce CDDP resistance. We have previously reported that human in vitro cell lines show different patterns of sensitivity to CDDP and 4 MeV X-irradiation. In these cells radiation sensitivity is related to high levels of expression of the c-raf-1 proto-oncogene. We thus predicted that cells sensitive to CDDP might show a different relationship to c-raf-1 expression. In addition, because cyclin D1 expression can be upregulated by the myc or ras oncogenes, we also chose to study putative relationships between cyclin D1 protein levels and intrinsic cellular sensitivity to CDDP and γ-irradiation. We report that in the 16 human cell lines which we have studied, high cyclin D1 expression is related to CDDP resistance but has no relationship with radiation responsiveness, whereas high c-raf-1 expression, although related to radiosensitivity has no relationship with CDDP responsiveness. © 1996 Wiley-Liss, Inc.

We compared apoptosis in mouse thymocytes following exposure to low doses of high linear energy transfer (LET), 62.5-MeV (p-->Be+) fast neutrons and low LET, 4-MeV photons by flow cytometric analysis of hypodiploid cells. The incidence of apoptotic cell death rose steeply at very low radiation doses reaching a plateau of 3 Gy. Both the time course and the radiation dose-response curves were similar for high and low LET radiation modalities. The relative biological effectiveness (RBE) of 1.0 for apoptosis in the mouse thymocyte system contrasts with the much higher value typically seen in many classical systems of clonogenic cell survival and tissue response. This difference suggests that while radiation-induced apoptosis may contribute significantly to loss of susceptible cells at doses of < or = 2 Gy, it appears to have a questionable role in determining the relative intrinsic radiosensitivity of mammalian cells to high and low LET irradiation at clinically relevant levels of cell kill.

This paper describes the development and structure of an Electronic Study Guide for Oncology (LETSGO) for undergraduate medical students. LETSGO is aimed at clinical students learning about cancer. The subject of the guide is breast cancer and learning objectives cover structure and function, behavioural science, public health and epidemiology and professional and personal values. LETSGO is designed to follow the steps used in problem-based learning. The student is encouraged to carry out individual brainstorming around cases with the issues identified acting as the first step in an educational audit loop. Clear definition of prior knowledge is available by way of interactive features, and hyper-text links to core text and diagrams (including microscopic sections) precede definition of both broad aims and objectives for the module and specific objectives for assessment purposes. Core knowledge is available via hyper-text links. Assessment has three components: open ended questions asking for free text responses linking to 'model' answers; extended matching items linking to 'model' answers and providing peer-referenced feedback as a bar-chart distribution, and an educational audit loop referring back to the original issues identified at the beginning of the package in brainstorming. Clear mapping throughout the guide is a major feature and the student's progress is clearly displayed at each stage of the guide. The program provides dynamic access to the student's existing knowledge base and stimulates new learning based on the student's own learning needs.

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 (T(pot)) 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 T(pot) or LI or with DNA ploidy.Conclusion: 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 a values for neutrons and photons is compared to the reciprocal of the a 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.

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.

Following conflicting results from Hammersmith and Edinburgh, the 62.5 MeV (p-->Be+) Douglas cyclotron was installed at Clatterbridge in order to carry out further studies with fast neutrons. Several features were incorporated into the study design to achieve as unbiased as possible a comparison between 62.5 MeV neutrons and conventional 8 MV x-ray therapy. Interim analysis of 151 patients in the pelvic study in the autumn of 1989 revealed a trend towards a worse survival in the neutron therapy group which soon became significant, leading to study termination in February 1990. The reasons for this diminished survival were unclear; with no significant difference in morbidity. Although the incidence of metastases was initially higher in the neutron patients than the photon patients this difference was not sufficient to explain the survival difference. Considerable morbidity would be expected from photon therapy using the same fractionation as was used in the neutron arm of the trial. If further neutron therapy at this energy is planned consideration should be given to the use of smaller fractions.

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.

The intrinsic sensitivity to 4 MeV photons, and 62.5 MeV (p-->Be+) neutrons has been examined in a panel of 11 cultured human cell lines exhibiting a wide spectrum of inherent cisplatinum sensitivity. Irrespective of whether cellular sensitivities to these therapeutic agents were compared at the 10% survival level, relative to the initial portion of the cell survival curves, or to their relative rank order of response, there were no significant correlations between inherent cisplatinum sensitivity and sensitivity to either 4 MeV photon, or 62.5 MeV neutron irradiation. This data raises the possibility that the previously reported decreased radiosensitivity of human tumour cell lines with acquired cisplatinum resistance may be due to the induction of cellular processes which confer resistance to both cisplatinum and ionising radiation, rather than the selection of innately cisplatinum-resistant cells, which are collaterally radioresistant.

Purpose: To determine the relative sensitivity to cis-platinum, 4 MeV photons and 62.5 MeV (p-->Be+) neutrons in five human tumor cell lines, and their cis-platinum resistant variants.Methods and Materials: The degree of cross-resistance of five human in-vitro cell lines to photons or fast neutrons was analysed for both cisplatinum-sensitive and resistant variants.Results: The development of acquired cis-platinum resistance conferred collateral resistance to 62.5 MeV (p - Be+) neutrons in all five cell lines, but did not consistently decrease the photon sensitivity of these same cells.Conclusion: The reduction in photon and neutron sensitivity following the development of acquired cis-platinum resistance may possibly be regulated by different mechanisms. The reduction in neutron sensitivity was primarily due to a 1.3-1.7 fold reduction in the magnitude of the initial slope (alpha), which was independent of the degree of platinum resistance induced, suggesting a non-stochiometric relationship between the mechanisms responsible for acquired cis-platinum, and 62.5 MeV (p-->Be+) neutron resistance.

The clinical role of fast neutron therapy has been limited by excessive late normal tissue damage. A pilot study of accelerated fractionation of fast neutron therapy was performed, based on the rationale that this should result in an increase in the response in acute reacting tissues (normal and malignant), with no change in late damage and a consequent increase in the therapeutic ratio. Further accelerated fractionation should improve the local control of rapidly proliferating tumour, without the potential problem of inadequate reoxygenation inherent in accelerated photon schedules. 6 or 12 fractions of 62 MeV (p-Be) neutrons were given over 12 days to 27 sites in 23 patients with locally advanced tumours. With a dose reduction of 12% (18 Gy), acceptable skin and oral mucosa early reactions were obtained. A larger dose reduction (15%) was required at pelvic sites. The incidence of late EORTC/RTOG grade 4 toxicity was 46%. The overall response rate was 76% with a complete response rate of 16%. For locally advanced breast cancer, the complete response rate was 9%, which compares unfavourably with previous results with conventional neutron fractionation schedules. The combination of a low overall complete response rate and excessive late normal tissue toxicity suggests that accelerated fractionation of fast neutrons does not lead to an improvement in the therapeutic ratio, and that late normal tissue damage will continue to be dose limiting.

In vitro studies have suggested that elevated levels of the thiol glutathione (GSH) may be associated with acquired alkylating agent resistance, but there is currently little data on the relationship between elevated GSH and glutathione S-transferase levels and clinical alkylating agent resistance. In this study, GSH and glutathione S-transferase levels have been determined in 23 human ovarian tumor samples obtained prior to the onset of combination chemotherapy, and in 23 samples obtained after the development of acquired chemoresistance. GSH levels were 10-fold greater in human ovarian tumor cells obtained after alkylating agent resistance developed, than in biopsy samples obtained prior to treatment. No significant changes in the expression of total glutathione S-transferases were seen in relation to prior drug exposure.

The inherent sensitivity of 20 human cell lines to the 62.5 MeV(p----Be+) clinical neutron beam at Clatterbridge, UK, has been assessed and compared to their sensitivity to 4 MeV photons. The survival curves of the cell lines following neutron irradiation were curvilinear, and the inherent neutron sensitivity varied by 4.5 fold (0.1 survival level) between the extreme values, in the cell lines studied. There was a strong correlation between the sensitivity of these human cells to photon and neutron irradiation. It was concluded that should these in vitro patterns occur in the clinic, the 4-fold variation in RBE and inherent sensitivity to neutrons could result in overall lower local control rates following fast neutron therapy than might be anticipated. It suggests the need for the development of predictive assays as a potential means of selecting tumours most appropriate for neutron therapy.