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Background: Normal adult mammalian cells can respond to oncogenic somatic mutations by committing suicide through a well-described, energy dependent process termed apoptosis. Cancer cells avoid oncogene promoted apoptosis. Oncogenic somatic mutations are widely acknowledged to be the cause of the relentless unconstrained cell proliferation which characterises cancer. But how does the normal cell with the very first oncogenic mutation survive to proliferate without undergoing apoptosis?, New Findings: The phenomena of malignant transformation by somatic mutation, apoptosis, aneuploidy, aerobic glycolysis and Cdk4 upregulation in carcinogenesis have each been extensively discussed separately in the literature but an overview explaining how they may be linked at the initiation of the cancer process has not previously proposed., Conclusion: A hypothesis is presented to explain how in addition to the initial oncogenic mutation, the expression of certain key normal genes is, counter-intuitively, also required for successful malignant transformation from a normal cell to a cancer cell. The hypothesis provides an explanation for how the cyclic amphiphilic peptide HILR-056, derived from peptides with homology to a hexapeptide in the C-terminal region of Cdk4, kill cancer cells but not normal cell by necrosis rather than apoptosis., (© 2023 The Author. Cancer Reports published by Wiley Periodicals LLC.)

Background: Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor., Results: A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP). An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy., Conclusion: These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with a companion diagnostic biomarker and a putative functional site for kinase-unrelated activities of Cdk4.

Background: Although the term theranostics has been coined only fairly recently, attempts to relate the level of biomarkers to therapeutic response in the oncology clinic go back several decades. After a long period in which a limited number of individual theranostic molecular biomarkers gained general clinical acceptance, extremely powerful genomic and proteomic technologies have now emerged., Methods: These technologies, reviewed here, promise a potential revolution in our ability to predict therapeutic response in cancer, and by so doing, guide new anticancer drugs more successfully into clinical oncology practice. A full understanding of the detailed molecular nature of clinical cancer is, however, still evolving. The need for appropriate models of the highly complex disease, against which we are attempting to direct effective therapy more accurately, is also addressed. These should include an understanding of genomic and proteomic heterogeneity, genetic instability and systems biology models of cancer that take into account recent demonstrations of the vastly increased mutational state of the average clinical cancer as compared with the normal cell(s) from which it arose., Conclusion: The way forward in theranostics is, arguably, less dependent on further improvements in the already powerful genomic and proteomic technologies themselves than on our improved understanding of how we should apply them to the complex reality of the average clinical cancer.

Despite major advances in the molecular biology of the cancer cell over the past two decades, the great majority of patients are still treated by conventional cytotoxic drugs. The chemotherapy regimens employed frequently include platinating agents, taxanes, intercalating agents and topoisomerase inhibitors. Attempts to predict the therapeutic efficacy of such drugs by molecular profiling (theranostics) have up to the present time had limited success. Genes responsible for the control of cell division, senescence and apoptosis whose normal functions become corrupted during carcinogenesis, might potentially play a part in determining chemotherapeutic response. Here we have examined the relationships between the chemoresponsiveness of 18 human in vitro cancer cell lines and proteomic expression of Ras, cyclins B1 and D1 and cyclin-dependent kinases Cdk1 and Cdk4. When all 18 cell lines were examined as a single group, proteomic expression did not provide any helpful theranostic predictors. Clear relationships between proteomic expression and drug efficacy emerged, however, when Ras, cyclin B1, cyclin D1, Cdk1 and Cdk4 were examined separately in p53 wild-type and p53 mutant cell subsets. We suggest that the theranostic relationships we have detected in vitro may have potential relevance in vivo and should prompt clinical theranostic studies which take account of p53 mutational status.

The majority of targets for rational anti-cancer drug development available at present have been defined by studies of early carcinogenesis. In this situation, mutations resulting in altered expression or structure of gene products, in the context of a relatively normal diploid background, provide the cell with a growth advantage. In contrast to cells studied in early carcinogenesis, however, profound chromosomal abnormalities and widespread disruption of gene expression can be found in the cells of typical cancers seen in the clinic. In addition tumour heterogeneity and genetic instability, in clinical cancers, can result in variable and elusive drug targets. The severe degree of genetic damage, seen in the clinical situation, would be expected to sufficiently disrupt the function of cell-signalling pathways, such that the highly complex and co-ordinated process of cell division, which occurs in normal cells, could no longer be effectively accomplished in the clinical cancer cell. It may therefore be relevant to consider whether certain Critical Normal Genes must be retained throughout the life of the progressively evolving cancer cell in order to enable it to continue to divide successfully. The regulatory roles of the products of such Critical Normal Genes would be expected to become particularly important in the chaotic molecular environment, which finally occurs in the typical clinical cancer cell. Here remaining Critical Normal Gene Products may be required to perform novel functions to maintain cellular integrity. Because the function of such gene products would be essential for continued cancer cell proliferation, they would not be expected to be subject to tumour heterogeneity or genetic instability as loss of function of a gene product essential to cell proliferation would inevitably result in the death of that cell. Perhaps such remaining Critical Normal Gene Products, with adapted functions critical to continued cancer cell proliferation, whose disruption may only have minimal effects within the multiple signalling pathways of normal cells and which are likely to be unaffected by tumour heterogeneity and genetic instability, could provide particularly relevant targets for anti-cancer drug development.

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 gamma-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., (Copyright 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).

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-diamminedichloroplatinum (CDDP) resistance. However, transfection of c-myc or v-H-ras and activation of protein kinase C (PKC), which contributes to the RAF-1, 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 gamma-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.

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.

The mechanism by which ultraviolet radiation induces melanogenesis in epidermal melanocytes is unknown. Previous observations that in cultured human melanocytes 1-oleoyl-2-acetylglycerol augmented both basal and ultraviolet radiation-induced melanogenesis, suggested that the responses were mediated via protein kinase C. However, paradoxically the phorbol ester TPA was without effect. Therefore, the present study has examined the involvement of protein kinase C in melanogenesis. Analysis of the isozyme profile of human melanocytes revealed the presence of protein kinase C alpha, beta I, epsilon and zeta but not the isozyme eta. Following exposure to 500 nM TPA for 24 hours, isozymes alpha, beta I and epsilon were downregulated, but zeta was unaffected. Similar isozyme profiles were observed in S91 and SKMEL3 melanoma cells. The melanogenic responses to 1-oleoyl-2-acetylglycerol and ultraviolet radiation were unaffected by inhibition of protein kinase C with Ro31-8220, or ablation by downregulation with 500 nM TPA, in human melanocytes and melanoma cells. 1-Oleoyl-2-acetylglycerol had no effect on protein kinase C activity in human melanocytes, as measured by rapid phosphorylation of the 80 kDa protein myristoylated alanine-rich C kinase substrate (MARCKS). Ultraviolet radiation induced a small increase in MARCKS protein phosphorylation but this effect was inhibited by pretreatment for 24 hours with 500 nM TPA, which had no effect on ultraviolet-induced melanogenesis. Overall, these findings indicate that 1-oleoyl-2-acetylglycerol and ultraviolet radiation activate melanogenesis via protein kinase C-independent pathways.

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.

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.

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.

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.

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.

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 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.

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.

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.

The melphalan resistant variant of the human ovarian OAW42 tumour cell line has previously been shown to be collaterally resistant to photon irradiation, but not to fast neutrons. In the present study, the "in vitro" photon and neutron radiosensitivity of human ovarian OAW42 tumour cells with acquired resistance to cis-platinum has been studied, to determine whether a similar pattern of cross-resistance exists between cis-platinum and these ionising radiations. Analysis of SF2 values suggests that resistance to cis-platinum conferred a 3-fold decrease in sensitivity to photons, primarily attributable to a 5-fold decrease in the magnitude of the initial slope (alpha). Depletion of GSH by BSO restored the magnitude of alpha to a value similar to that of the parental line. However, cis-platinum resistant OAW42/CP cells, in contrast to melphalan resistant cells, were 1.5-fold more resistant to "fast" neutrons (assessed at D0.1 survival level) than the parental OAW42 cell line. The mechanism for the collateral resistance between cis-platinum, and both photons and neutrons remains to be determined, but although GSH levels may be directly, or indirectly involved in the collateral resistance to photons, they would appear not to involved with the mechanisms responsible for collateral neutron resistance, in the cis-platinum resistant human ovarian cell lines used in this study.

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.

Acquired resistance to cis-platinum and melphalan, in the human ovarian OAW42 tumor cell line, respectively, conferred a 3- and 1.5-fold decrease in photon sensitivity. Analysis of cell survival curves by the linear quadratic equation showed an accompanying 5- and 2-fold reduction in the magnitude of the initial slope (alpha). Treatment with the GSH depleting agent BSO restored the magnitude of alpha to a value similar to that of the parental line without evidence of dose modification in the high-dose region of the cell survival curve. This in conjunction with failure of alteration in GSH levels to affect parental OAW2 sensitivity and of the SER of BSO to reflect GSH levels suggest a possible GSH independent mechanism of action for BSO. If similar patterns occur in the clinic, the possibility exists of circumventing collateral resistance between chemotherapeutic agents and ionizing radiation, provided that tumor thiol levels can be preferentially depleted.

High linear energy transfer (LET) fast neutrons for the local control of advanced head and neck tumours are currently being evaluated at several centres. Fast neutrons are believed to produce more direct, and less OH mediated damage than photons, and consequently be less affected by intracellular thiol levels. Chemoresistant tumours with elevated thiol levels may therefore be more effectively controlled by fast neutron therapy than by photons. The "in vitro" radiation response of melphalan sensitive and resistant human ovarian tumour cell lines has demonstrated that melphalan resistance confers a 1.5-fold level of cross-resistance to photons, primarily attributable to a 2-fold decrease in the alpha component in the resistant OAW42/MER cell line. Pretreatment of the melphalan-resistant line with the thiol depleting agent buthionine sulphoximine (BSO) restored the magnitude of alpha to a value similar to that in the chemosensitive cell line. The survival curves of these cell lines following neutron irradiation were near exponential, with similar values of alpha. This study has demonstrated that melphalan resistant tumour cells are cross-resistant to photon irradiation, but not to fast neutrons. The mechanism of cross-resistance has yet to be determined, but glutathione (GSH) appears to be involved.

The lymphocyte subpopulations of peripheral blood of normal lambs and lambs experimentally infected with bovine respiratory syncytial virus (RSV) were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with bovine RSV was characterized by a significant rise in SBU-T8+ (CD8+ or cytotoxic) T cells and a significant reduction in SBU-T4+ (CD4+ or helper) T cells and B (LCA p220+) lymphocytes (P less than 0.05). The helper/suppressor (CD4/CD8) ratio was reduced from 3.91 on the day of experimental infection to 1.13 on 10 days after experimental infection (P less than 0.001). The total number of SBU-T4+ (CD4+) and B cells returned to pre-inoculation values 14 days after experimental infection but the helper/suppressor ratio remained depressed up to 21 days post-inoculation.

The HT29R human colonic adenocarcinoma cell line grows as locally invasive, mucin-secreting tumours in immunosuppressed mice with a doubling time of 6 days. These tumours may be disaggregated to give single-cell suspensions with plating efficiencies of 25-45% in technically simple in vivo-in vitro cell-survival assays. The effect of maximum tolerated doses of 5-fluorouracil, melphalan and cyclo-phosphamide on in situ growth is only slight. In vivo-in vitro cell-survival assays phosphamide on in situ growth is only slight. In vivo-in vitro cell-survival assays are consistent with these in situ results. The relative ease of experimental manipulation and the high clonogenic efficiency of this tumour make it a useful addition to human tumour xenograft models.

Seventy-seven patients with small cell lung cancer were entered on a protocol comprising induction chemotherapy with cyclophosphamide 1 g m-2, adriamycin 40 mg m-2 and vincristine 1.4 mg m-2 (CAV) every 21 days for four to six cycles. The overall response rate was 72.8%. Twenty-six patients achieving complete remission received intensification with two further cycles of ifosfamide 5 g m-2, mesna 8 g m-2, methotrexate 30 mg m-2 and etoposide 100 mg m-2 per day for 3 days (IME). Six of the 15 patients in partial remission following CAV achieved a further remission on IME (response rate 40%). Median survival in the limited disease group was 11 months compared with 7 months in the extensive disease patients and four patients are alive at more than 2 yr follow up. There was no significant prolongation of the median survival (11 months) seen in those patients in complete remission who had negative second bronchoscopy examination. This sequential six drug regime produces high response rates in small cell lung cancer, and there is evidence of lack of cross-resistance between CAV and IME.

A model is proposed which relates reproductive death of cells caused by radiation to loss of chromatin at cell division. This loss of chromatin can occur through chromosomal deletions or through the formation of asymmetrical chromosomal exchanges. It is proposed that smaller doses of radiation produce fewer chromatin breaks, which are more likely to be accurately repaired, compared with larger doses. Consequently, smaller doses of radiation are less efficient in causing cell death, leading to a shoulder on the cell survival curve. Experimental evidence supports this model, and the fit between the derived formula and experimental cell survival curves is good. The derived formula approximates to the linear-quadratic equation at low doses of radiation.

We describe the use of 31P NMR spectroscopy in the study of metabolic changes related to hypoxia in cultured human tumor cells in vitro. The 31P NMR spectrum can easily distinguish between metabolically active cells, metabolically inactive "dormant" cells, and necrotic cells. A crucial observation was that of the ability of the "dormant" cells to resume active metabolism on incubation with oxygen after long periods of hypoxia.

The HT29R colonic adenocarcinoma xenograft has been shown to be rich in the enzyme beta-glucuronidase. Experiments in rodent systems have demonstrated a marked anti-tumour effect of the drug aniline mustard (AM) on tumours with high levels of this enzyme (e.g. the plasmacytomas PC5 and PC6). We have found that AM is no more effective than its analogue paramethyl aniline mustard (PMAM) or other alkylating agents against the HT29R xenograft. Amongst the possible explanations for this may be: (1) The wide shoulder on the cell-survival curve shown for exposure to alkylating agents of HT29R in vivo. (2) Lack of correlation between physiological availability of beta-glucuronidase and the high levels measured by the standard assay. (3) Increased beta-glucuronidase levels in host mouse marrow, making the latter potentially more susceptible to AM damage.

31 patients with intermediate and high-grade non-Hodgkin lymphomas were treated by a six-drug alternating regime comprising four cycles of 200 mg/m2 i.v. methotrexate on days 8, 15, 28 and 35, 50 mg/m2 i.v. Adriamycin on day 1, 40 mg/m2 oral prednisolone on days 1-7 and 21-27, 120 mg/m2 i.v. etoposide on days 21-23, 600 mg/m2 i.v. cyclophosphamide on day 21 and 1.4 mg/m2 i.v. vincristine on day 1 (MAPECO). In all, 3 patients had stage I disease, 12 stage II, 6 stage III and 10 stage IV. Of 28 evaluable patients, 19 were complete responders (68%) and 9 were partial responders (32%); at 2 years, the actuarial relapse-free survival of the 19 patients achieving complete remission is 80%, and 5 patients remain in complete remission at 3 years. This is a preliminary report of an effective intensive regime with acceptable toxicity.

Eighteen consecutive cases of encephalopathy occurring after ifosfamide/mesna chemotherapy were prospectively assessed. No relationship was found with tumour type or chemotherapy response. Onset was from 12 to 146 (mean 46) h after the start of the infusion and median duration was 3 days (range 1-12). In two patients recovery was incomplete. A confusional state and agitation were the major clinical features. Plasma potassium fell from a mean of 4.12 mmol/l before chemotherapy to 2.94 mmol/l at the onset of encephalopathy (P less than 0.001) with plasma potassium less than 3.0 mmol/l in 10 patients. Duration of hypokalaemia was not related to duration of encephalopathy. Median survival following encephalopathy was 25 days. The incidence of encephalopathy in 82 patients treated on two protocols was 11% and the sensitivity of a published nomogram was 18%. It is concluded that ifosfamide/mesna encephalopathy is a serious complication which may be irreversible and remains difficult to predict.

Human immunoglobulin-secreting hybridomas have been produced by fusion of the LICR-LON-HMy2 line with lymph node lymphocytes from patients with malignant melanoma. Immunoglobulin production as measured by reverse haemolytic plaque assay was greater for hybridomas than for the parent HMy2 line. Pre-incubation of unseparated cultures from lymph nodes with pokeweed mitogen resulted in more successful fusions with a greater yield of hybridomas. The hybrids did not, however, have any greater immunoglobulin production than did those from lymphocytes not pre-incubated with pokeweed mitogen. Using lymph node cells from a patient with malignant melanoma of the cervical region, a consistent pattern of increased immunoglobulin binding to three human melanoma cell lines in contrast to a human colonic adenocarcinoma and a human skin-derived fibroblast line was demonstrated.

The feasibility, effectiveness and toxicity of a new four drug intensive combination chemotherapy regime has been assessed in 36 patients with inoperable non-small cell lung cancer. Three cycles of adriamycin, vindesine, ifosfamide and cisplatin were given to 36 patients followed by sequential radiotherapy to 25 patients. The overall response rate to chemotherapy was 33%, and median progression free interval was 6 months. Median survival was 8.5 months. Grade 4 WHO neutropenia was seen in 12 patients, there were three episodes of neutropenic fever and one death in which toxicity may have been contributory. Radiotherapy was not associated with serious toxicity. This study demonstrates that a significant response rate can be achieved with short intensive chemotherapy in unresectable non-small cell lung cancer and that it is feasible to combine this with radiotherapy.

Preliminary results are reviewed on the outcome of patients treated within two randomized studies with either p(60) + Be-neutrons or photons. Since April 1987 67 patients have been treated of which twelve have been included in a randomized study on head and neck cancer and 40 on pelvic cancer. The clinical treatment planning is presented in detail and discussed. The results presented are considered to be very preliminary, so that no attempt has been made to analyse and discuss them in detail.

We have investigated the potential for using anti-sense non-ionic methylphosphonate oligonucleotide analogues to study the relationship between oncogene expression and maintenance of the transformed phenotype in malignant cells. Our results confirmed that the methylphosphonates are resistant to biochemical degradation and are devoid of non-specific toxicity towards cultured human HT29 cells. At low temperature (less than 5 degrees C) both N-ras anti-sense and nonsense analogue 9-mers formed 1:1 complexes in solution with an N-ras sense phosphodiester oligodeoxynucleotide 20-mer, but these were largely dissociated at 25 degrees C. Only a fraction (10-20%) of the anti-sense molecules formed stable sequence specific hybrids (Tm 34 degrees C) with the 20-mer. The biological activity of the oligonucleotide analogues was tested in cell culture at 37 degrees C using T15 cells, a line of NIH 3T3 cells transfected with multiple copies of the human N-ras oncogene under control of the glucocorticoid inducible MMTV promoter. On balance the N-ras anti-sense methylphosphonate 9-mer (20-80 microM) had no effect on these cells. In only one of five experiments was an apparent reduction in dexamethasone-induced p21N-ras protein accumulation observed in the presence of the oligonucleotide analogue. Also without effect was an anti-sense 20-mer consisting of a phosphodiester sequence bounded by two methylphosphonate linkages at each end (25-50 microM in culture media; 4.8 microM by microinjection). We conclude from these experiments that, in order to achieve pronounced effects on oncogene expression, it may be necessary to use longer anti-sense methylphosphonate chains, affinity purified for their ability to hybridize with the target sequences.

The plasma pharmacokinetics of etoposide administered at a dose of 600 mg/m2/day over 3 consecutive days to 19 patients are described. High performance liquid chromatography with ultraviolet detection was used to determine the etoposide levels. The plasma concentration time curve showed a biphasic decay. When given as a 6-h infusion of 600 mg/m2 on each of 3 consecutive days, the peak plasma levels (PPL) were 45.6 +/- SD 10.5 micrograms/ml, and the area under the curve (AUC) values were 1379.6 +/- SD 332.4 micrograms/ml/h. In 2 patients a second cycle 28 days later as a 72-h infusion at a total dose of 1,800 mg/m2 was given, and in these cases the mean PPL was 30 micrograms/ml compared to 63 micrograms/ml for the 6-h infusion in the same patients, and the mean AUC was 1,239 micrograms/ml/h compared to 1,952.5 micrograms/ml/h. There was no correlation with age, sex, the duration of or the severity of the neutropenia. It is concluded that high dose etoposide has higher PPL and improved concentration X time values when given as 3 consecutive daily infusions 18 h apart than as a continuous 72 h infusion.