Biomedical subjects

H M Warenius

Publications and source records attributed to H M Warenius.

At least 19 recordsLinked to original sources

PubMed2000

Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity.

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.

Amino Acid Sequence↗

PubMed2000

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.

Animals↗

PubMed1998

Late G1 accumulation after 2 Gy of gamma-irradiation is related to endogenous Raf-1 protein expression and intrinsic radiosensitivity in human 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).

Blotting, Western↗

PubMed1996

Sensitivity to cis-diamminedichloroplatinum in human cancer cells is related to expression of cyclin D1 but not c-raf-1 protein.

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.

Antineoplastic Agents↗

PubMed1996

Exit from G2 phase after 2 Gy gamma irradiation is faster in radiosensitive human cells with high expression of the RAF1 proto-oncogene.

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).

Cell Line↗

PubMed1995

RBE of fast neutrons for apoptosis in mouse thymocytes.

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.

Animals↗

PubMed1995

An electronic study guide for problem-based learning.

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.

Adult↗

PubMed1994

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.

Cell Survival↗

PubMed1994

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.

Energy Transfer↗

PubMed1994

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.

Blotting, Western↗

PubMed1994

Ultraviolet radiation-induced melanogenesis in human melanocytes. Effects of modulating protein kinase C.

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.

1-Methyl-3-isobutylxanthine↗

PubMed1994

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.

Cell Division↗

PubMed1994

Fast neutron therapy. The UK experience.

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.

Fast Neutrons↗

PubMed1993

The differential induction of collateral resistance to 62.5 MeV (p-->Be+) neutrons and 4 MeV photons by exposure to cis-platinum.

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.

Cell Survival↗

PubMed1993

De novo cisplatinum resistance does not influence cellular radiosensitivity.

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.

Cell Line↗

PubMed1992

Collateral resistance to photon and neutron irradiation is associated with acquired cis-platinum resistance in human ovarian tumour cells.

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.

Cell Survival↗

PubMed1992

BSO-induced reduction of glutathione levels increases the cellular radiosensitivity of drug-resistant human tumor cells.

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.

Buthionine Sulfoximine↗

PubMed1992

Cellular glutathione (GSH) and glutathione S-transferase (GST) activity in human ovarian tumor biopsies following exposure to alkylating agents.

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.

Antineoplastic Combined Chemotherapy Protocols↗