GB2334579A - Sensitivity of cancer cells to anti-cancer agents involving measurement of properties of signal transduction factors - Google Patents

Abstract

A method for the measurement of the sensitivity of a cancer cell to an anti-cancer agent comprises assay of a sample for the mutational status, expression and/or function of a negative signal transduction factor (NSTF), and the mutational status, expression and/or function of a positive signal transduction factor (PSTF), provided that assay of the radiosensitivity of wild-type p53 cancer cells for Raf-1 protein is effected using an antibody thereto. The NSTF may be a factor which inhibits or arrests the cell cycle, causes cells to withdraw from the cell cycle, and/or causes apoptosis or other cell death thereby inhibiting cell division (especially a suppressor), or may be p53, p21 or a PSTF inhibitor (preferably a Raf-1 inhibitor, a cyclin D1 inhibitor or a cyclin-dependent kinase inhibitor). The PSTF may be a factor which stimulates cells to enter the cell cycle, initiates and/or effects DNA synthesis, and/or controls the passage of cells through the cell cycle, preferably an oncogene, a proto-oncogene, a gene which inhibits and/or controls cell cycle division, or a cell surface receptor (especially Raf-1 protein, cyclin D1 protein, or a cyclin-dependent kinase, particularly CDK1 or CDK4). The anti-cancer agent may be ionising radiation, a molecular anti-cancer agent (preferably a chemotherapeutic agent) or a biological cancer therapy agent. Kits, for performing the methods, are described.

Description

TREATING CANCER The present application concerns methods of selecting the most appropriate therapy for patients suffering from cancer. The application is particularly concerned with measuring the resistance of cancer cells to anti-cancer agents.

Although radiotherapy and chemotherapy have been responsible for curing many people of cancer in the latter half of this century, there still remain a large number of tumours which either show little response to treatment, or respond initially only to recur later. In particular, women treated for ovarian cancer with platinating agents often show encouraging initial responses to chemotherapy (which in the UK usually involves the use of cisdiamminedichloroplatinum as the drug of first choice), but by 5 years after diagnosis, 2/3 of them have succumbed to their disease. Similarly lung cancer patients may respond favourably to combination chemotherapy regimens containing cisdiamminedichloroplatinum (CDDP) at the outset of treatment but very few experience long term survival. A better understanding of the mechanisms underlying the responsiveness of cancers to CDDP, could help predict which patients are most likely to benefit from CDDP or whether alternative cytotoxic agents such as Taxol or different therapies such as radiotherapy might be appropriate. Understanding treatment response mechanisms also holds the possibility of selectively modulating these mechanisms to improve the treatment of human cancer using CDDP.

It has become increasingly apparent that certain oncogenes and tumour suppressor genes may not only be implicated in carcinogenesis, but can also influence the sensitivity of malignant cells to therapeutic agents. Attempts have therefore been made to use these and other genes to try and predict the therapeutic response of human cancer to the presently available treatment modalities such as radiotherapy and/or cytotoxic chemotherapy. Research up to the present time, however, has generally attempted to only examine the expression of single tumour related genes as methods of predicting therapeutic response. Research in the public domain has suggested that mutations in the p53 tumour suppressor gene, which can be found in around 50% of common cancers such as those of the breast, lung and ovary, are associated with resistance to treatment with cytotoxic drugs or radiation. Despite a considerable body of work, however, there are at present no successful clinical tests by which the detection of mutations in the p53 gene alone can be used to predict with an acceptable degree of certainty whether or not a patient's cancer is likely to respond to chemotherapy with, for example, platinating agents or the newer cytotoxic agents such as Taxol.

The expression of single genes alone on the response of human cancer cell lines to treatment with cytotoxic drugs such as cisdiamminedichloro-platinum (CDDT) has been studied in human in vitro cell lines because these present a model system relevant to the response of human cancer in the clinic. In particular, they exhibit the range of sensitivities to cytotoxic drugs and ionising radiation usually encountered in the clinic. Discoveries in human in vitro cell lines, therefore, have a strong possibility of being able to be translated into clinically useful tests for how well cancers may be expected to respond to treatment. The protein products of the cyclin D1 and B1 genes and their respective cyclin-dependent kinase partners CDK4 and CDK1 have been studied. Cyclin Dl and CDK4 control the progress of cells through the cell cycle checkpoint between G1 and S-phase (the phase of DNA synthesis). Cyclin B1 and CDK1 control the cell cycle checkpoint just before mitosis. The expression of cyclin D1 protein in a series of 16 human cancer cell lines has been shown to be related to their intrinsic resistance to the cytotxic drug cis-diamminedichloroplatinum (II) (CDDP) (Warenius et al., 1996). Cyclin Dl protein levels, however, showed no relationship with radiosensitivity, another treatment modality. The relationship between cyclin D1 and CDDP resistance is not, however, strong enough on its own to provide the basis of clinically useful predictive assays.

Thus, there are no indicators that measuring the mutational status or levels of expression of the protein products of single oncogenes, proto-oncogenes or tumour suppressor genes in human cancer cells would be able to provide the basis of a reliable clinical test for whether clinical tumours were likely to respond to treatment with chemotherapeutic agents, including platination agents and CDDP.

Although radiotherapy has been responsible for curing many people of cancer in the latter half of this century, there still remain a large number of tumours which either show little response to treatment, or respond initially only to recur later. A better understanding of the mechanisms underlying the responsiveness of cancers to radiotherapy, could help predict which patients are most likely to benefit from radiotherapy, and also holds the possibility of selectively modulating these mechanisms to improve the treatment of human cancer using radiotherapy.

The molecular basis of intrinsic radiosensitivity has been under investigation for many years. A considerable body of research has focussed on the degree of DNA damage and its subsequent repair as reflected in the incidence of double strand breaks (dsbs) in the DNA (Kelland et al, 1988; Schwartz et al, 1991), the residual damage remaining in the DNA after cellular rejoining of dsbs (Nunez et al, 1995; Whitaker et al, 1995), and the fidelity of DNA repair (Powell & McMillan, 1994). In addition to DNA damage, however, it has become increasingly apparent that certain oncogenes and tumour suppressor genes may not only be implicated in carcinogenesis, but can also influence the sensitivity of malignant cells to ionizing radiation.

As a result of this growing evidence of the role of oncogenes and tumour suppressor genes in the sensitivity of malignant cells to therapeutic agents, attempts have been made to use these and other genes to try and predict the therapeutic response of human cancer to the presently available treatment modalities such as radiotherapy and/or cytotoxic chemotherapy. Research up to the present time, however, has generally attempted to only examine the expression of single tumour related genes as methods of predicting therapeutic response. When investigating the relationship between expression of a chosen gene and intrinsic radiosensitivity, consideration has not necessarily been given as to whether other candidate genes than the one selected for study might also have an affect on the outcome of experiments.

This consideration could be particularly relevant in human cancers, the majority of which exhibit disorders of expression of multiple oncogenes and suppressor genes.

Research into the role of individual genes has focussed on a number of cell cycle genes and signal transduction genes.

Transfection of normal cell lines with dominant oncogenes such as myc and ras (McKenna et al, 1991) has resulted in increased radioresistance even in the absence of detectable changes in the rate of dsb induction (Iliakis et al, 1990). Several other dominant oncogenes including c-fms, v-sis, v-erb-B, v-abl, v-src, v-cot (Fitzgerald et al 1990, Suzuki et al, 1992, Shimm et al, 1992) and c-Raf (Kasid et al, 1989, Pirollo et al, 1989) have also been reported to modulate cellular radiosensitivity in mammalian cells. The potential relevance of these findings to clinical radiotherapy has been emphasised by observations that high levels of Raf-1 (the normal protein product of the c-Raf-1 proto-oncogene) are related to intrinsic radiosensitivity in human in-vitro cell lines (Warenius et al, 1994). However, these results are not sufficient alone to determine the sensitivity of a tumour to radiotherapy in a clinical assay.

An additional body of evidence indicates a positive relationship between mutation in the p53 tumour suppressor gene and increased cellular radioresistance in both rodent and human tumour cells (Fan et al, 1994, Radford 1994, Zhen et al, 1995, Xia et al, 1995, Lee and Bernstein 1993) and in normal cells transfected with mutant p53 (mp53) genes (Pardo et al, 1994, Bristow et al, 1994, Kawashima et al, 1995). Research in the public domain has suggested that mutations in the p53 tumour suppressor gene, which can be found in around 50% of common cancers such as those of the breast, lung and ovary, are associated with resistance to treatment with cytotoxic drugs or radiation. Despite a considerable body of work, however, there are at present no successful clinical tests by which the detection of mutations in the p53 gene alone can be used to predict with an acceptable degree of certainty whether or not a patient's cancer is likely to respond to radiotherapy. A wide disparity of results in clinico-pathological studies comparing tumour response and p53 status leads to the conclusion that at the present time p53 mutation or the over expression of p53 protein are not sufficient alone to predict whether or not a human cancer is likely to respond to radiotherapy.

A number of reports suggest that oncogenes and suppressor genes may modulate intrinsic radiosensitivity by their influence on the progress of irradiated cells through radiation-induced blocks at cell cycle checkpoints. G1/S delay, mediated by p53 following exposure to ionizing radiation has been implicated as an important measure of cell cycle perturbation which correlates with relative radiation sensitivity (Kastan et al, 1991, McIlwrath et al, 1994; Siles et al, 1996). Also the expression of dominant oncogenes such as myc and ras (McKenna et al, 1991) or SV40 (Su & Little, 1993) has been shown to induce both radioresistance and a concomitant increase in post-radiation delay at the G2/M checkpoint. It has also been shown that the protein product of the normal c-Raf-l proto-oncogene was related to radiosensitivity in 19 human in-vitro cell lines (Warenius et al, 1994). Recently, it has further been shown that in 6 of the above 19 cell lines, the previously observed Raf1/radiosensitivity relationship was very strong and related to how rapidly cells exited from a radiation-induced block at the G2/M cell cycle checkpoint. Those radiosensitive human cancer cells with increased expression of the normal Raf-l protein exhibit more rapid exit from a G2/M block induced by 2Gy of radiation than radioresistant cells with low expression of Raf-1 (Warenius et al, 1996). High expression of the Raf-1 protein product of the normal c-Raf proto-oncogene is related to radiosensitivity but has no relationship with resistance to CDDP.

The relationship between Raf-l and radiosensitivity is not, however, strong enough on its own to provide the basis of clinically useful predictive assays. The same is true of other attempts to correlate the effects of single genes to the success of therapies.

Unfortunately, little is known about whether, or how, oncogenes and suppressor genes may interact to influence the radiosensitivity phenotype of human cancer cells. However, transfection experiments using cells from other mammals, such as REF (rat embryo fibroblasts), have demonstrated greater increases in radioresistance in cells expressing dominant plus co-operating oncogenes than expressing the single dominant oncogenes alone (McKenna et al, 1990, Su & Little 1992, Pirollo et al, 1993).

Similarly, radioresistance induced in REF cells by transfection with multiply integrated mutant p53-pro193 alleles was much greater when the mutant p53 gene was co-transfected with H-ras (Bristow et al, 1994).

Thus, at the present time there are no indicators that measuring the mutational status or levels of expression of the protein products of single oncogenes, proto-oncogenes or tumour suppressor genes in human cancer cells would be able to provide the basis of a reliable clinical test for whether clinical tumours were likely to respond to drug and/or radiation treatment.

It has been shown more recently (Warenius et al, 1994, 1996) that measuring Raf-l protein in the context of wild-type p53 provides a correlation which could possibly provide the basis of a predictive assay for radiosensitivity. This relationship was demonstrated by measuring Raf-1 protein using quantitative Western blotting. Western blotting is, however, expensive, time consuming and labourious. Furthermore, it requires large numbers of cells. It is thus impractical as a routine clinical test.

A clinical assay must be capable of measuring protein levels in individual cells, rather than tn homogenates of a million or more cells as used in Western blotting. It is also important to be able to distinguish Raf protein expression in tumour cells from that in normal cells. This requires the ability to gate out cells on the basis that they are diploid rather than aneuploid in flow cytometry assays, or the ability to measure Raf protein in individual cells that can be observed histologically on tissue sections where morphological criteria enable regions of tumour to be distinguished from connective tissue, blood vessels infiltrating white blood cells, or area of necrosis.

Unfortunately all available antibodies against Raf cross-react with an irrelevant epitope on a 48 kD molecule, when examined on Western blots (see Fig. 2). Raf-l is a 72-74 kD molecule and can thus be distinguished and separately measured on Western blotting. Cellular assays for Raf-l such as flow cytometry or immunocytochemistry would not, however, be able to distinguish the correct 72-74 kD molecule from the irrelevant 48 kD molecule.

The 48 kD protein is unlikely to be a fragment of the 72 kD Raf proto-oncogene because the 48 kD protein is much more abundant than the 72 kD protein on Western blotting.

Invention: An object of the present invention is to solve the above problems. Accordingly, this invention provides a method for measuring the sensitivity of a cancer cell to an anti-cancer agent, which method comprises testing a sample for a correlation between the mutational status expression and/or function of a negative signal transduction factor (NSTF), and the mutational status expression and/or function of a positive signal transduction factor (PSTF) , wherein the method does not comprise measuring the radiosensitivity of wild-type p53 cancer cells by testing a sample comprising wild-type p53 cells or an extract therefrom for the abundance of Raf-l protein other than by employing an antibody specific to Raf-l protein.

In the context of this invention a factor includes any gene, molecule, component or product, and in particular such factors which are contained in cells.

This invention also provides a kit for measuring the sensitivity of a cancer cell to an anti-cancer agent, which kit comprises means for testing a sample for the mutational status, expression, and/or function of a negative signal transduction factor (NSTF) and the mutational status, expression, and/or function of a positive signal transduction factor (PSTF) The present invention will be described in further detail by way of example only with reference to the accompanying drawings, in which: Figure 1A shows the relationship between the level of cyclin D1 protein and relative resistance to CDDP in mutant p53 cell lines; Figure 1B shows the corresponding relationship in wild-type p53 cell lines; Figure 2 shows a Western blot demonstrating the range of Raf-l protein levels per total cellular protein, in particular the relative abundance of the 74 kD and 48 kD proteins, in the following 17 human in vitro cell lines: 1. KB, oral epidermoid carcinoma 2. HT29, adenocarcinoma, colon 2. MGH-U1, bladder carcinoma 4. HRT18, adenocarcinoma, rectum 5. A431, squamous carcinoma vulva 6. NCTC 2544, skin fibroblasts 7. COR L23, large cell lung carcinoma 8. SK-MEL3, melanoma 9. AT5BIVA, ataxia telangiectasia fibroblast 10. OAW42, ovarian carcinoma 11. I407, embryonic intestinal epithelium 12. 2780, ovarian carcinoma 13. HEP-2, squamous carcinoma 14. HX142, neuroblastoma 15. RT112, bladder carcinoma 16. HeLa, squamous carcinoma 17. NCTC 2544 for a sample protein loading of 100 pg/50p1 on a 7.5 % gel, the primary antisera being URP-2653 monoclonal against Raf-l at a dilution of 1/750; Figure 3A shows the relationship between radiosensitivity measured as SF2 (log surviving fraction at 2 Gy) and Raf-l abundance in wild-type p53 cell lines; Figure 3B shows the relationship between radiosensitivity measured as SF2 and Raf-l abundance in mutant p53 cell lines; Figure 4A shows the relationship between radiosensitivity measured as SF2 and Raf-l abundance in cell lines in which p21 protein levels are elevated; Figure 4B shows the relationship between radiosensitivity measured as SF2 and Raf-l abundance in cell lines in which p21 protein levels are not elevated; Figure 5A shows the relationship between the level of cyclin D1 protein and relative resistance to CDDP in cell lines in which p21 protein levels are elevated; and Figure 5B shows the relationship between the level of cyclin D1 protein and relative resistance to CDDP in cell lines in which p21 protein levels are not elevated.

A first embodiment In a preferred embodiment, this invention provides methods of predicting whether human cancer cells are likely to respond to anticancer therapy agents (chemotherapeutic agents, such as platination agents e.g. CDDP) by contemporaneously measuring the properties of two or more cancer-related genes. Moreover the corelationship between certain independently expressed cancer genes identified in this invention also provide previously undescribed targets against which to potentially direct therapy that is more cancer specific.

Thus, this embodiment specifically deals with measuring the levels of Cyclin D1 protein, in cells whose p53 mutational status has been determined (e.g. by DNA sequencing) to determine the resistance of a tumour to (for example) cisdiamminedichloroplatinum (CDDP). High cyclin D1 levels or high cyclin Dl expression together with p53 mutation is strongly associated with resistance to CDDP in human cancer cells.

Human cancer cell lines with a combination of p53 mutation and high levels of expression of the cyclin D1 protein are resistant to CDDP. This finding carries important clinical possibilities with regard to providing a potentially new parameter for predictive assays for CDDP responsiveness or a new target for modulating CDDP responsiveness.

The high correlation of p53 mutations with high cyclin Dl levels or cyclin D1 over-expression also provides a potential target for drug development. Efforts are being made to develop drugs against mutant forms of p53 and independently, against cyclin D1. Such drugs are likely to be more effective when used together to treat cancers with the above p53 mutations and cyclin D1 overexpression. Such drugs might also be used in combination with other agents such as CDDP as potentiators of its effectiveness.

A Dual Parameter Test for CDDP Resistance Using p53 Mutational Status and Cyclin D1 Protein Expression Fig. 1A shows that in p53 mutant human cell lines there is a strong relationship between the level of cyclin D1 protein and relative resistance to CDDP as measured by the D0.1 values. The implication is that human cancer cells with p53 mutations and high levels of cyclin D1 protein are unlikely to respond to CDDP and alternative therapy such as Taxol should be considered. It is possible that Taxol sensitivity is not influenced by a combination of p53 mutation and cyclin D1 protein overexpression. The cyclin Dl/p53 mutation test may also detect resistance to other cytotoxic drugs such as etoposide but ionising radiation does not have any relationship to cyclin D1 over-expression in p53 mutant cells. Thus the test will indicate situations where radiation might be a viable alternative to CDDP, or whether other cytotoxic agents might be more appropriate.

A clinical test may be developed for CDDP sensitivity based on the dual measurement of Cyclin D1 protein expression and the presence of mutations in the p53 gene. Cyclin D1 protein is typically measured by Western blotting or immunocytochemistry in a research environment but for diagnostic purposes cheaper and more rapid methods are desirable.

The determination of the mutational status of p53 can be effected by sequencing the genomic locus bearing the gene from the patient or by sequencing the expressed mRNA after conversion to cDNA.

Various nucleic acid sequencing methodologies are available at present, all of which are appropriate for use with this diagnostic assay. The typical method would be based on incorporation of terminating nucleotides into polymerase generated copies of a template, using the method of Sanger et al, 1977. Many alternatives have arisen recently including adaptor sequencing (PCT/US95/12678), ligation based sequencing (PCT/US96/05245), sequencing by hybridisation (A.D. Mirzabekov, TIBTech 12: 27 - 32, 1994) to list a few. Various methods for testing for specific mutations are known in the art, such as the TaqMan assay, oligonucleotide ligase assays, single strand conformational polymorphisms and assays based on hybridisation of template nucleic acids to oligonucleotide arrays.

Because cyclin D1 is a relatively short lived protein under cyclical transcriptional control, it is likely that mRNA levels for cyclin D1 will follow the same pattern as the cyclin D1 protein and show a similar strong relationship to CDDP resistance. This would make it possible to carry out a functional assay for resistance to CDDP by extracting mRNA from tumour samples and using this to determine the relative abundance of cyclin D1 mRNA and to detect mutations in the p53 mRNA.

Oligonucleotide Arrays Determination of mRNA levels can be effected in a number of ways.

One can readily convert poly-A bearing mRNA to cDNA using reverse transcription - a method is described in the example illustrating this invention. Reverse Transcriptase PCR (RTPCR) methods allow the quantity of single RNAs to be determined, but with a relatively low level of accuracy. Arrays of oligonucleotides are a relatively novel approach to nucleic acid analysis, allowing mutation analysis, sequencing by hybridisation and mRNA expression analysis. Methods of construction of such arrays have been developed, ( see for example: A.C. Pease et al. Proc. Natl.

Acad. Sci. USA. 91, 5022 - 5026, 1994; U. Maskos and E.M.

Southern, Nucleic Acids Research 21, 2269 - 2270, 1993; E.M.

Southern et al, Nucleic Acids Research 22, 1368 - 1373, 1994) and further methods are envisaged. Arrays that measure expression levels of mRNAs and detect mutations in those RNAs are being developed and these offer an attractive embodiment of the diagnostic test proposed by this invention.

Immunocytochemi s try An alternative aspect of this embodiment would measure Cyclin D1 protein levels by immunocytochemistry using confocal laser fluorescence microscopy. Preferably a scanning system would be used such as those described in PCT/US91/09217, PCT/NL/00081 and PCT/US95/01886. Additionally, it is desirable that the microscopy system should also be able to analyse multiple fluorescent dyes.

Antibodies against mutant forms of p53 would be labelled with one dye, an antibody against cyclin D1 (sc-6281, Santa Cruz Biotechnology, CA) would be labelled with a second dye whilst a third DNA binding dye could be used to select for aneuploid cells. DNA binding dyes such as Hoechst 33258 dye, which binds AT-rich DNA or Chromomycin A,, which binds GC-rich DNA, would be appropriate. At present not all mutant forms of p53 can be detected using antibodies, although antibodies exist against a number of known mutant forms of the p53 protein. A diagnostic test might comprise the steps of: o Extracting a biopsy of the tumour from a patient. o Optionally micro-dissecting that material to separate normal tissue from tumour material. o Preparing the biopsy material for microscopy which includes the steps of: - Labelling the biopsy material with the above fluorescently labelled antibody probes against Cyclin D1. The biopsy material may also, Optionally be labelled with antibody probes against p53 mutant proteins and with a DNA binding dye.

- Separating the labelled cells from unbound labelled probes. o Placing the labelled biopsy material in a scanning confocal microscope to count cells that: - Over-express or show elevated levels of cyclin D1, i.e. are labelled with at least a threshold quantity of antibody against cyclin D1.

- Optionally, express mutant forms of p53, i.e. are labelled with at least the threshold quantity of antibodies against p53 mutants. Alternatively, p53 mutational status might be determined by analysis of the mRNA or genomic DNA as discussed above.

- Optionally, have chromosomal amplifications as detected by the intensity of fluorescence from DNA binding fluorescent dyes.

Fluorescence Activated Cell Sorting A further embodiment of the diagnostic test could exploit Fluorescence Activated Cell Sorting (FACS). A FACS instrument separates cells in a suspension in a manner dependant on the cells being labelled with a fluorescent marker. A typical FACS device operates as follows. Cells in a suspension travelling in single file are passed through a vibrating nozzle which causes the formation of droplets containing a single cell or none at all. The droplets pass through a laser beam. Fluorescence excited from each individual cell in its droplet by the laser is measured. After the detector the stream of cells in suspension pass through an electrostatic collar which gives the droplets a surface charge. The cell carrying droplets are given a positive or negative charge. If the drop contains a cell that fluoresces with an intensity above a particular threshold, the drop gets a charge of one polarity. Unlabelled cells get a charge of the opposite polarity. The charged droplets are then deflected by an electric field and depending on their surface charge are directed into separate containers and are counted. Droplets that contain more than one cell scatter light more than individual cells which is readily detected and so these are left uncharged and enter a third disposal container. Multi-channel fluorescent detection devices have been constructed that can separate cells on the basis of labelling with multiple different fluorescent labels.

These have multiple lasers which can excite fluorescence at different frequencies and the detector will detect different emmission frequencies. A three label system would be appropriate for this test. The same labelled probes as those described above for use in a confocal scanning fluorescence microscope would be appropriate. A diagnostic test might comprise the steps of: o Extracting a biopsy of the tumour from a patient. o Optionally micro-dissecting that material to separate normal tissue from tumour material. o Disrupting intracellular adhesion to form a single cell suspension. o Labelling the suspended cells with the above fluorescently labelled probes against cyclin D1. The biopsy material may also, optionally be labelled with antibody probes against p53 mutant proteins and with a DNA binding dye. o Separating the labelled cells from unbound labelled probes. o Passing the labelled cell suspension through a FACS device to count cells that: - Over-express or show elevated levels of cyclin D1, i.e. are labelled with the anti-cyclin D1 antibody above a threshold for 'normal' expression.

- Optionally, express a mutant form of p53, i.e. are labelled with at least a threshold quantity of antibody against mutant forms of p53.

- Optionally, have chromosomal amplifications as detected by the intensity of fluorescence from DNA binding fluorescent dyes.

Modulation of Cyclin D1 Expression in p53 Mutant Human Cancers At present many attempts are being made to develop drugs which inhibit cyclin D1. As this molecule has a vital function in controlling the progress of normal cells through the 'start' component of the G1/S checkpoint such inhibitors would be likely to be extremely non-selective and very toxic to normal cells. The more specific relationship of cyclin D1 to resistance to CDDP in p53 mutant human cancer cells described here provides a much more defined target for novel therapeutic agents which could potentially used in conjunction with CDDP itself an agent with a proven track record of curing many (though by no means all) cancers. This app range of human in-vitro cell lines by measuring the expression of target genes and/or determining their mutational status and correlating these parameters to cell line sensitivity to cytotoxic agents. This procedure has identified genes relevant to clinical responsiveness to CDDP. Discoveries in human in vitro cell lines, such as those leading to this invention, therefore, have a strong possibility of being able to be translated into clinically useful tests for how well cancers may be expected to respond to treatment. The body of work that has been carried out to measure the clonogenic cell survival of a wide range of human in-vitro cell lines of different histology after exposure to CDDP is described below.

Materials and Methods Cell lines and clonogenic cell survival assays The growth characteristics clonogenic assay procedures of the 12 human in vitro cell lines used in this analysis have already been reported (Warenius et al 1994). The cell lines are listed, with their histological classification in Table 1. All are well established; many having been growing in vitro for several years.

Cell lines were either donations or purchased by our laboratories. On receipt all were grown for 5 passages to provide sufficient cells for batch storage in liquid nitrogen. During this period contamination was excluded by at least one passage in antibiotic free medium and mycoplasma testing was carried out on all lines. For clonogenic assays, cells were taken from a designated primary liquid nitrogen batch and grown for 3-6 passages until there were sufficient well-growing cells. Further batches from these cells were frozen in liquid nitrogen. Cells were routinely maintained in DMEM medium except RT112 and H322, which were grown in RPMI1640 and MGHU-1 which were grown in Ham's F12 medium. All lines were supplemented with 10% heat-inactivated fetal calf serum (HIFCS) In order to assay CDDP sensitivity 102 - 105 cells were plated in 3 ml of Ham's F12 medium supplemented with 10% FCS in 6 well plates and incubated at 372 C in an atmosphere of 5% CO2 for 8 hours. Dilutions of 0.02 - 2.0 yg/ml from a 1 mg/ml stock solution of CDDP (light protected) were then made and 1 ml of the appropriate dilution were added to each plate to give a final volume of 4 ml. The plates were then incubated at 370 C in an atmosphere of 5% CO2 in darkness for 14 days in the presence of the CDDP. The medium was then removed, the cells were fixed in 70 W ethanol and stained with 10% Giemsa and colonies of > 100 cells counted. One 6 well plate was used for each drug dilution.

The data points from all the assays were pooled to provide means and SEMs as shown in Figures 3a and 3b. Where no SEMs are visible, the range of the SEM lies within the magnitude of the pooled data point. A minimum of 3 separate clonogenic assays with 6 points/drug dose/assay were necessary for each cell line. CDDP cell survival was determined at the 10% clonogenic cell survival level (D0.1) by interpolation of the fitted regression curve.

Identification of mutations in the p53 gene by PCR and DNA sequencing Material for PCR and DNA sequencing of p53 and Western blotting for cyclin-D1 protein, was obtained from the same liquid nitrogen batches used to provide cells for clonogenic cell survival data.

Cells were grown for up to three passages prior to being subjected to the following procedures: Nucleic Acid Isolation RNA and genomic DNA were prepared from the cell lines described here by the guanidinium isothiocyanate CsC1 gradient method (Chirgwin et al, 1979, Barraclough et al, 1987). Briefly, the cells were collected in ice-cold phosphate-buffered saline (PBS) and homogenised in guanidinium isothiocyanate buffer (4M guanidinium isothiocyanate, 50mM Tris pH 7.5, 25mM EDTA pH 8.0, 0.5k (w/v) sodium lauryl sarcosine and 8% (v/v) 2-mercaptoethanol added just prior to use. The homogenate was cleared by centrifugation at 8,000 rpm for 10 mins at 4 C (SS34 rotor, Sorvall RC-5B centrifuge) and the RNA pelleted by centrifugation of the homogenate through a cushion of 5.7M caesium chloride/0.lM EDTA at 32,000 rpm for 20hr at 20OC (TST 41.14 rotor, Kontron Centrikon T20 60 ultracentrifuge). The pellet of RNA was redissolved in 0.1% (w/v) SDS and precipitated with ethanol overnight at -20OC before quantitation.

Polymerase Chain Reaction, cDNA synthesis and nucleotide sequencing PCR (for exons 2-8 and for exons 9-11) was performed on DNA and RNA extracted from the 18 human carcinoma cell lines. Each exon was then examined by DNA sequencing. PCR Primers were designed flanking each exon and synthesised on an Applied Biosystems 381A DNA synthesiser. Each exon was amplified separately with the exceptions of exons 2 and 3 which were amplified as a unit, and exons 9, 10 and 11 which were amplified together by reverse transcription polymerase chain reaction (RTPCR) . The following primers were used: Exon 2/3 sense 5'-CCC ACT TTT CCT CTT GCA GC-3 Exon 2/3 antisense 5'-AGC'CCA ACC CTT GTC CTT AC-3' Exon 4 sense 5'-CTG CTC TTT TCA CCC ATC TA-3' Exon 4 antisense 5'-GCA TTG AAG TCT CAT GGA AG-3' Exon 5 sense 5'-TGT TCA CTT GTG CCC TGA CT-3' Exon 5 antisense 5'-CAG CCC TGT CGT CTC TCC AG-3' Exon 6 sense 5'-GCC TCT GAT TCC TCA CTG AT-3' Exon 6 antisense 5'-TTA ACC CCT CCT CCC AGA GA-3' Exon 7 sense 5'-ACT GGC CTC ATC TTG GGC CT-3' Exon 7 antisense 5'-TGT GCA GGG TGG CAA GTG GC-3' Exon 8 sense 5'-T ATC CTG AGT AGT GG-3' Exon 8 antisense 5'-T GCT TGC TTA CCT CG-3' Exon 9/10/11 sense 5'-AGA AAG GGG AGC CTC ACC-AC-3' Exon 9/10/11 antisense 5'-CTG ACG CAC ACC TAT TGC AA-3' Genomic DNA was digested with EcoR1 and precipitated with ethanol and resuspended in 50y1 of water (Sigma) before being subjected to PCR amplification. The DNA (lAg) was amplified in 50cm1 PCR reactions containing 20 pmoles of each primer. A 'hot start' PCR protocol was used with the dNTP's and Taq enzyme initially separated from the rest of the reaction components on a wax cushion. The reactions were placed in a pre-heated PCR block at 95"C for 2 minutes before undergoing thirty cycles of denaturation (30s at 95 C), annealing (30s at 602C for exons 2-3, 4 and 6; 65C for exons 5 and 8; 67OC for exon 7; and 68oC for exon 9-11) and extension (1 min at 72C). The PCR products were checked on a 0.8k (w/v) agarose gel before being purified using a Wizard minicolumn (Promega), and used directly in sequencing reactions. cDNA synthesis and RTPCR Complementary DNA was synthesised from approximately 5yg of total RNA using oligo (dT) as a primer. Total RNA (5 g), human placental ribonuclease inhibitor (HPRI) 20U and lpg oligo (dT) were heated at 70OC for 10 minutes, chilled on ice, added to lx first strand buffer (50mM Tris-HC1, pH 8.3, 75mM potassium chloride and 3mM magnesium chloride), 0.01M DTT, dNTPs (0.5mM for each deoxyribonucleoside triphosphate), 400U of Superscript Reverse Transcriptase (Gibco) and incubated at 37"C for 1 hour.

PCR for exons 9 to 11 was carried out using 5y1 of the above incubation in a 50p1 of PCR reaction as described in the previous section.

Nucleotide Sequencing Sequencing primers (10 pmoles) were radioactively labelled at their 5' ends with '2P-ATP (45pCi) at 37C for 30 min in a reaction containing T4 Polynucleotide Kinase (PNK) (9.7U, Pharmacia) and lx T4 PNK buffer (lOmM Tris-acetate, 10mM magnesium acetate and 50mM potassium acetate). The primers used were identical to those employed in the PCR reactions except for exon 5 for which a separate sense sequencing primer was designed as follows:- 5'-TAC TCC CCT GCC CTC-3'. Sequencing was carried out by the dideoxynucleotide enzymatic method (Sanger et al, 1977), using the fmol DNA Sequencing System (Promega) . Any putative sequence mutations identified were confirmed by additional sequencing of the exon in the antisense direction as well as by carrying out a repeat PCR and sequencing of the cell line.

Western Blotting for Cyclin D1 Two independent Western blottings with lysates for each cell line loaded in pairs on each gel were carried out. Standard conditions were used for the preparation of cells for lysates for Western blotting on each of the 16 cell lines; 107 cells were grown in 162 cm' tissue culture flasks (Costar Ltd., High Wycombe, Bucks) until they were pre-confluent but still growing exponentially as confirmed by flow cytometry. Cells were then removed by trypsinisation, resuspended in complete medium + 10% FCS and washed 3 times by serial centrifugation and resuspension in PBS without serum. 1-3 x 108 viable cells were then pelletted by centrifugation and resuspended at 3x107 cells per ml of lysate buffer (Stock solution: 10% SDS 10ml., 0.5M Tris pH 6.8, glycerol 10 ml., Double distilled water 62 ml. To 10 ml. of stock solution were added 100 ml of 10 mM Leupeptin + 10 ml 100 mM PMSF).

Protein estimations were performed and the final concentration of the lysates adjusted to 300 jig total cellular protein per 100 jil. To measure cyclin Dl protein, 150jig of total cellular protein in 50 jil of lysate buffer was added per lane well to a 7.5% Laemmli separating gel and electrophoresis carried out at 16"C using 60V over 16 hours and a constant current of 500mA. Blots were transferred to nitrocellulose at 22"C over 16 hours using to a semi-dry blotting apparatus (Biorad, Richmond, CA), incubated with the a mouse IgG1 monoclonal antibody to mammalian cyclins (G124-259.5, Pharmingen) and then incubated with rabbit antimouse conjugated antibodies (Dako, UK) at 1/1000 and developed in alkaline phophatase buffer containing Nitroblue Tetrazolium and 5-Bromo-4-Chloro-3-Indoyl Phosphate, (Sigma, Poole, Dorset, UK) (SOmg/ml in dimethylformamide) for 1 hr at room temperature in darkness. Colour development was arrested with double distilled water1 and the blots were dried flat. Cyclins were clearly resolved as distinct bands, cyclin D1 having the lowest mobility.

Quantitation of the protein product of the cyclin D1 gene was carried out by measurement of optical density on a Schimadzu scanning densitometer with tungsten light and expressed as O.D. units per 150 jig of total cellular protein. Titration curves obtained by loading different amounts of total cellular protein have previously shown that linear relationships for optical density (O.D.) could be obtained over the range found for cyclin D1 protein across the cell lines (Warenius et al 1994, Browning 1997) . In order to compare different cyclin D1 protein levels between the cell lines, the mean O.D. value for all the lines was calculated and the relative O.D. for cyclin D1 protein in each individual cell line was normalised to the mean O.D. and multiplied by an arbitrary value of 5.0.

Results Mutations were found in mRNA expressed from the p53 gene in 7 of the 12 cell lines. The mutations identified in the cell lines described here were in exons 5-8 which are known to contain the majority of p53 mutations (Hollstein et al, 1991). All these mutations have been previously described apart from the nonsense mutation identified in codon 166 of the RPMI7951 line. This along with the G to T transversion in codon 298 of H417 did not lie within the most highly conserved region of the p53 gene. In the OAW42 ovarian carcinoma cell line the single base missense mutation from CGA to CGG was silent, so that the mutant triplet still coded for the same amino acid (Arg) as is present in wild type p53 (wtp53) protein (Fig.lA) . A normal p53 protein was thus expressed in 6 of the 12 cell lines. The mRNA of the other 6 cell lines coded for abnormal p53 protein. RPMI7951 and H417 possessed stop mutations resulting in 165 and 297 amino acid truncated proteins respectively. COLO320 and H322 independently exhibited a missense G:C to A:T mutation at the same site resulting in an amino-acid substitution from Arg to Tryp. RT112 and HT29/5 also had mutations coding for changes in Arg (to Gly and His respectively!. COLO32Q, H322 and RT112 were homozygous for p53 mutations. The other three mutant lines showed evidence of retention of heterozygosity (as shown for H417 in Fig.lB).

HT29/5 and RPMI7951 both expressed small amounts of wild-type p53 mRNA though H417 expressed relatively high levels.

The relationship between cyclin D1 levels and CDDP sensitivity was examined for all 12 cell lines and then independently in the wtp53 and mp53 cells. The ranges of cyclin-D1 protein levels in wtp53 cells and mp53 cells overlapped (3.33 - 10.39 and 3.58 8.46 respectively). Only in p53 mutant cells was a useful correlation found between cyclin D1 protein levels and resistance to CDDP.

Conclusions In mutant p53 cell lines, the higher the cyclin D1 levels, the more likely it is that the cells are resistant to CDDP (Fig. 1A).

A second embodiment It is a further object of this invention to provide an assay which can be used as a clinical assay to predict whether cancer cells are likely to respond to radiotherapy. It is also an object of this invention to provide an antibody against Raf-l which is specific for that protein, to facilitate cheaper diagnostic tests. A further object of the invention is to provide a method, using the antibody, for predicting whether cancer cells are likely to respond to radiotherapy by contemporaneously measuring the properties of two or more cancer-related genes.

Accordingly, the present invention provides a method for measuring the radiosensitivity of wild-type p53 cancer cells, which method comprises testing a sample comprising wild-type p53 cells or an extract therefrom for the abundance of Raf-l protein, wherein the testing is carried out using an antibody specific to Raf-l protein.

The present invention also provides a method for producing an antibody specific to Raf-l protein, which antibody does not cross-react with a 48 kD protein co-present in cells containing Raf-l protein, which method comprises forming a peptide which comprises or forms part of an epitope on the Raf-l protein that is not present on the 48 kD protein, and preparing an antibody against the peptide. The invention provides a further method for producing an antibody specific to Raf-l protein, which antibody does not cross-react with a 48 kD protein co-present in cells containing Raf-l protein, which further method comprises immunising an animal with Raf-l protein and an antibody specific to the 48 kD protein so as to mask potential epitopic sites on Raf-l protein which are also present on the 48 kD protein, and obtaining an antibody against the masked Raf-l protein.

Furthermore, the present invention provides a kit for measuring the radiosensitivity of wild-type p53 cancer cells, which kit comprises a means for testing for the abundance of Raf-l protein and a means for identifying wild-type p53 cells.

This embodiment specifically deals with measuring the levels of the Raf-l protein product of the C-Raf-l proto-oncogene, in cells whose p53 mutational status has been identified, preferably by DNA sequencing, to determine the radiosensitivity of the tumour and consequently whether radiotherapy is an appropriate treatment for the patient. In a background of unmutated p53, the higher the level of Raf-l expression the greater the sensitivity of the tumour to ionising radiation.

Whilst the mechanisms explaining the observed relationships between radiosensitivity, cell cycle progress and the functions of Raf-1 and p53 remain obscure, the strong relationship between Raf-l and radiosensitivity in human cancer cells expressing wildtype p53, permits the development of a dual parameter Raf-l/p53 test for clinical radiosensitivity.

Figure 3A shows that in p53 wild-type cell lines, the higher the Raf-l protein, the more radiosensitive the cells are as measured by log surviving fraction at 2 Gy (SF2). On the other hand, Figure 3B shows that in the presence of p53 mutations there is little or no relationship between Raf-l levels and radiosensitivity.

The clinical test requires determination of the mutational status of p53 as wild-type, in conjunction with measuring the level of Raf-l expression in biopsy material from tumours in patients. The determination of the mutational status of p53 can be effected by sequencing the genomic locus bearing the gene from the patient or by sequencing the expressed mRNA after conversion to cDNA.

Various nucleic acid sequencing methodologies are available at present, all of which are appropriate for use with this diagnostic assay. The typical method would be based on incorporation of terminating nucleotides into polymerase generated copies of a template, using the method of Sanger et al, 1977. Many alternatives have arisen recently including adaptor sequencing (PCT/US95/12678), ligation based sequencing (PCT/US96/05245), sequencing by hybridisation to oligonucleotide arrays (A.D. Mirzabekov, TIBTech 12: 27-32, 1994) to list a few.

Various methods for testing for specific mutations exist such as the TaqMan assay or oligonucleotide ligase assays. However, these may not be entirely appropriate since the absence of known mutations may not necessarily imply that p53 in a tumour is in fact wild type.

Determination of the expression level of Raf-l is effected by measuring the abundance of the Raf-l protein. Raf-l protein levels can be measured by immunocytochemistry or flow cytometry (FCM). Previously there was a problem with the latter approach arising out of cross-reactivity of existing antibodies with non Raf-l proteins. Until the present, there were no available antibodies to Raf-l which did not also cross-react with a very abundant but irrelevant 48 kD protein on Western blotting.

Techniques such as immunocytochemistry or FCM would only give non-specific results. This necessitated some form of molecular separation, such as by electrophoresis in western blotting, to separate Raf-l (a 72-74 kD protein) from the irrelevant 48 kD species. Column chromatography techniques were appropriate, such as gel filtration or ion exchange chromatography. High Performance Liquid Chromatography or Capillary Electrophoresis were also usable as separation techniques. These could all be followed by an immunoassay. Other means of specific recognition may also have been conceivable, including the development of RNA aptamers to the Raf-l protein. However, all of these techniques are time consuming and expensive, making them inappropriate for a clinical test. The availability of an antibody specific to Raf-l allows diagnostic assays to be carried out without the need for separation.

The first method for producing the antibody according to the present invention required isolation and identification of the 48 kD cross-reacting epitope, so that its DNA and protein genetic sequence could be determined. This information was used to compare the 48 kD protein sequence with the full length Raf proto oncogene protein sequence to choose an epitope on the full length proto-oncogene protein that is not shared by the 48 kD protein.

This could be achieved using a cell line with high Raf-l and 48 kD protein levels, such as NCTC 2544 (see Fig. 1) - Quantities of lysate were produced and the 48 kD protein purified.

Purification could be achieved using immunoprecipitation or affinity purification with a monoclonal antibody produced by the inventors, or with a commercial anti-Raf-1 antibody, both of which have been shown on Western blotting to bind strongly to both the 72-74 kD and the 48 kD protein. The cells producing the monoclonal antibody were grown up in high yields for affinity chromatography as ascites in Balb/C mice. The antibody from the ascitic fluid was reacted with cyanogen bromide sephacryl and the antibody-sephacryl used to make an affinity column. The lysates from the NTCT cells were loaded onto the affinity column, nonspecific material was washed through and 48 kD and 72-74 kD molecules sharing the same epitope and bound to the antibody on the column were eluted at low pH. The immunoprecipitate or eluate from the affinity chromatography column was then concentrated, a protein estimation carried out and 150 jig per well was run on 10-20 adjacent wells in 10 W SDS poly-acrylamide gel electrophoresis with molecular markers. The 48 kD band was then excised and as long an amino acid sequence as possible was sequenced from the N-terminus (or alternatively from the Cterminus). Using the peptide sequence primers were prepared whose genetic sequences match the protein sequences (allowing for the degeneracy in the coding for certain amino acids) for the N (or C-) terminus. A series of PCRs was run until a 48 kD length of DNA was obtained. This was then sequenced. A sequence comparison between the 48 kD and the 72-74 kD full length Raf-l proto-oncogene enabled the rational design of synthetic peptides from potential epitopes on the full length 72-74 kD Raf protooncogene protein, which were not present on the 48 kD protein.

The unique synthetic peptides were used to prepare polyclonal and monoclonal antibodies which reacted against the Raf-1 protein, but not the 48 kD protein, these antibodies being valuable in flow or confocal microscopic cytometry and/or immunofluorescence or immunocytochemistry.

The second method for producing the antibody according to the present invention requires immunisation of an animal with Raf protein in addition to an antibody against the 48 kD protein. For example, either the 72-74 kD full length Raf-l protein (produced from the DNA sequence in an expression vector in bacteria) can be pre-incubated with previously available anti Raf-1 antibodies (which cross-react with the 48 kD protein) and the resulting immune complexes separated by centrifugation and then injected into an animal (such as a mouse), or the anti Raf-1 (anti 48 kD protein) antibodies and the full length 72-74 kD Raf-1 protein can be injected separately into the same animal. Polyclonal and monoclonal antibodies were prepared using the full length 72-74 kD Raf proto-oncogene protein as immunogen. The protein was produced by the recombinant Raf-1 gene in an expression vector.

The antibody against the 48 kD protein was a commercially available antibody. Its function was to cover potential epitopic sites on the Raf protein which cross-react with the 48 kD protein. Such a masked Raf protein immunogen more selectively stimulates the production of antibodies which recognise the full length 72-74 kD Raf proto-oncogene protein rather than the 48 kD protein.

A third embodiment A preferred embodiment of the present invention provides a method for measuring the resistance of a cancer cell to the cytotoxic effects of chemotherapeutic agents, which method comprises testing a sample comprising cells in which the p21 protein is elevated, or an extract therefrom, for the abundance of cyclin DI protein.

This embodiment also provides a method for measuring the radiosensitivity of a cancer cell, which method comprises testing a sample comprising cells in which p21 protein is not elevated, or an extract therefrom, for the abundance of Raf-1 protein.

Example 2 A number of cell lines were selected and their resistance to CDDP was tested in relation to their levels of cyclin D1 protein. The results for cell lines in which p21 protein levels were elevated were plotted on one graph, whilst the results for cell lines in which p21 protein levels were not elevated were plotted on a separate graph. In the case where p21 protein levels were elevated, the higher the cyclin D1 level, the greater the resistance to CDDP. This correlation allows a choice of treatment to be made. In the case where p21 protein levels were not elevated the correlation was not particularly marked and was insufficient for selecting an appropriate treatment (see Figure 5A and Figure 5B).

Example 3 A number of cell lines were selected and their radiosensitivity was tested in relation to their levels of Raf-l protein. The results for cell lines in which p21 protein levels were elevated were plotted on one graph, whilst the results for cell lines in which p21 protein levels were not elevated were plotted on a separate graph. In the case where p21 protein levels were not elevated, the higher the Raf-l level, the greater the radiosensitivity of the cells. This correlation allows a choice of treatment to be made. In the case where p21 protein levels were elevated the correlation was not particularly marked and was insufficient for selecting an appropriate treatment (see Figure 4A and Figure 4B).

References Barraclough et al, J. Cell Physiolog 131: 393 - 401, 1987.

Chirgwin et al, Biochemistry 18: 5294 - 5299, 1979.

Maskos and Southern, Nucleic Acids Research 21, 2269 - 2270, 1993.

Pease et al. Proc. Natl. Acad. Sci. USA. 91, 5022 - 5026, 1994.

Sanger et al, Proc. Natl. Acad. Sci. USA 74: 5463 - 5467, 1977.

Southern et al, Nucleic Acids Research 22, 1368 - 1373, 1994.

Warenius et al., Int.J.Cancer. 67: 224 - 231, 1996.

Bristow et al., Oncogene 9: 1527 - 1536, 1994.

Bristow et al., Radiotherapy and Oncology 40: 197 - 223, 1996.

P. W. G. Browning, "Proto-oncogene expression and intrinsic radiosensitivity, PhD Thesis, University of Liverpool, 1997.

Deacon et al, Radiotherapy and Oncology 2, 317 - 323, 1984.

Fan et al, Cancer Res. 54: 5824 -5830, 1994.

Fertil & Malaise, Int. J. Radiat. Oncol. Biol. Phys. 7: 621 629, 1981.

FitzGerald et al, Radiat. Res. 122: 44 - 52, 1990.

Hollstein et al, Science 253: 49 - 53, 1991.

Iliakis et al., Cancer Res. 50: 6575 - 6579, 1990.

Kasid et al. , Cancer Res. 49: 3396 - 3400, 1989.

Kastan et al, Cancer Res. 51: 6304 - 6311, 1991.

Kawashima et al., Int. J. Cancer 61: 76 - 79, 1995.

Kelland et al, Radiat. Res. 116: 526 - 538, 1988 Lee and Bernstein, Proc. Natl. Acad. Sci. USA 90: 5742 - 5746, 1993.

McIlwrath et al., Cancer Res. 54: 3718 - 3722, 1994.

McKenna et al., Cancer Res. 50: 97 - 102, 1990.

McKenna et al., Radiat. Res. 125: 283 - 287, 1991 Nunez et al, Br. J. Cancer 71: 311 - 316, 1995 Pardo et al., Radiat. Res. 140: 180 - 185, 1994 Pirollo et al., Int. J. Radiat. Biol. 55: 783 - 796, 1989 Pirollo et al., Radiat. Res. 135: 234 - 243, 1993.

Powell & McMillan, Int. J. Rad. Oncol. Biol. Phys., 29: 1035 1040, 1994.

Radford, Int. J. Radiat. Biol. 66: 557 - 560, 1994.

Sanger et al, Proc. Natl. Acad. Sci. USA 74: 5463 - 5467, 1977.

Schwartz et al., Int. J. Radiat. Biol. 59: 1314 - 1352, 1991.

Shimm et al., Radiat. Res. 129: 149 - 156, 1992.

Siles et al., Br. J. Cancer 73: 581 - 588, 1996.

Su & Little, Int. J. Radiat. Biol. 62: 461 - 468, 1992.

Su & Little, Radiat. Res. 133: 73 - 79, 1993.

Suzuki et al., Radiat. Res. 129: 157 - 162, 1992.

Warenius et al., Eur. J. Cancer 30, 369 - 375, 1994.

Warenius et al., Rad. Research 146,485 - 493, 1996.

Whitaker et al., Int. J. Radiat. Biol. 67: 7 - 18, 1995.

Xia et al., Cancer Res. 55: 12 - 15, 1995.

Zhen et Powell & McMillan, Int. J. Rad. Oncol. Biol. Phys., 29: 1035 1040, 1994.

Radford, Int. J. Radiat. Biol. 66: 557 - 560, 1994.

Sanger et al, Proc. Natl. Acad. Sci. USA 74: 5463 - 5467, 1977.

Schwartz et al., Int. J. Radiat. Biol. 59: 1314 - 1352, 1991.

Shimm et al., Radiat. Res. 129: 149 - 156, 1992.

Siles et al., Br. J. Cancer 73: 581 - 588, 1996.

Su & Little, Int. J. Radiat. Biol. 62: 461 - 468, 1992.

Su & Little, Radiat. Res. 133: 73 - 79, 1993.

Suzuki et al., Radiat. Res. 129: 157 - 162, 1992.

Warenius et al., Eur. J. Cancer 30, 369 - 375, 1994.

Warenius et al., Rad. Research 146,485 - 493, 1996.

Whitaker et al., Int. J. Radiat. Biol. 67: 7 - 18, 1995.

Xia et al., Cancer Res. 55: 12 - 15, 1995.

Zhen et al., Mut. Res. 346, 85 - 92, 1995.

Claims (38)

1. CLAIMS: 1. A method for measuring the sensitivity of a cancer cell to an anti-cancer agent, which method comprises testing a sample for the mutational status, expression, and/or function of a negative signal transduction factor (NSTF), and the mutational status, expression, and/or function of a positive signal transduction factor (PSTF) , wherein the method does not comprise measuring the radiosensitivity of wild-type p53 cancer cells by testing a sample comprising wild-type p53 cells or an extract therefrom for the abundance of Raf-l protein other than by employing an antibody specific to Raf-l protein.
2. 2. A method according to claim 1, wherein the NSTF is a factor which inhibits or arrests the cell cycle, causes cells to withdraw from the cell cycle1 and/or causes apoptosis or other cell death thereby inhibiting cell division.
3. 3. A method according to claim 1 or claim 2, wherein the NSTF is a suppressor.
4. 4. A method according to any preceding claim, wherein the NSTF is p53 or p21.
5. 5. A method according to claim 1 or claim 2, wherein the NSTF is a PSTF inhibitor.
6. 6. A method according to claim 5, wherein the PSTF inhibitor is a Raf-l inhibitor, a cyclin D1 inhibitor or a cyclin dependent kinase inhibitor.
7. 7. A method according to any preceding claim, wherein the PSTF is a factor which stimulates cells to enter the cell cycle, initiates and/or carries out DNA synthesis, and/or controls the passage of cells through the cell cycle.
8. 8. A method according to claim 7, wherein the PSTF is an oncogene, a proto-oncogene, a gene which inhibits and/or controls cell cycle division, or a cell surface receptor.
9. 9. A method according to claim 7 or claim 8, wherein the PSTF is Raf-l protein, cyclin D1 protein or a cyclin dependent kinase, such as CDK1 or CDK4.
10. 10. A method according to any preceding claim, wherein the anticancer agent is ionising radiation, or a molecular anti-cancer agent such as a chemotherapeutic agent or a biological cancer therapy agent.
11. 11. A method according to claim 1 for measuring the resistance of p53 mutant cancer cells to the cytotoxic effects of chemotherapeutic agents, which method comprises testing a sample comprising p53 mutant cells or an extract therefrom for the abundance of cyclin D1 protein.
12. 12. A method according to claim 1 for measuring the resistance of a cancer cell to the cytotoxic effects of chemotherapeutic agents, which method comprises testing a sample comprising cells in which the p21 protein is elevated, or an extract therefrom, for the abundance of cyclin D1 protein.
13. 13. A method according to any preceding claim, wherein the sample is extracted from a subject.
14. 14. A method according to any of claims 11-13, wherein the chemotherapeutic agent is a platinating agent.
15. 15. A method according to claim 14, wherein the platination agent is CDDP.
16. 16. A method according to claim l for measuring the radiosensitivity of wild-type p53 cancer cells, which method comprises testing a sample comprising wild-type p53 cells or an extract therefrom for the abundar,ce of Raf-1 protein, wherein the testing is carried out using an antibody specific to Rat-l protein.
17. 17. A method according to claim 16, wherein the antibody does not cross-react with a 48 kD protein co-present with the Raf-l protein in the sample.
18. 18. A method according to claim 17, wherein the antibody is obtainable by forming a peptide which comprises or forms part of an epitope on the Raf-1 protein, which epitope is not present on the 48 kD protein, and preparing the antibody against the peptide.
19. 19. A method according to claim 17, wherein the antibody is obtainable by immunising an animal with Raf-l protein and an antibody specific to the 48 kD protein so as to mask an epitopic site on Raf-1 protein that is also present on the 48 kD protein, and obtaining the antibody against the masked Raf-1 protein.
20. 20. A method according to claim 18 or claim 19, wherein the antibody is a monoclonal antibody.
21. 21. A method according to any of claims 16-20, wherein the antibody is a labelled antibody.
22. 22. A method according to claim 21, wherein the label is a fluorescent label.
23. 23. A method according to claim 1 for measuring the radiosensitivity of a cancer cell, which method comprises testing a sample comprising cells in which p21 protein is not elevated, or an extract therefrom, for the abundance of Raf-l protein.
24. 24. A method according to any preceding claim, wherein the sample is a sample of cells.
25. 25. A method according to claim 24, wherein the testing is carried out by performing a cell count.
26. 26. A method according to claim 25, wherein the cell count is performed using multi-parameter flow cytometry.
27. 27. A method according to claim 25, wherein the cell count is performed using scanning confocal microscopy.
28. 28. A method according to claim 25, wherein the cell count is performed using fluorescence activated cell sorting.
29. 29. A method according to any of claims 25-28, wherein the sample of cells is micro-dissected prior to performing the cell count, to separate normal tissue from tumour tissue.
30. 30. A method according to any of claims 25-29, wherein prior to performing the cell count, intracellular adhesion in the sample is disrupted, to form a single cell suspension.
31. 31. A kit for measuring the sensitivity of a cancer cell to an anti-cancer agent, which kit comprises a means for testing a sample for the mutational status, expression, and/or function of a negative signal transduction factor (NSTF), and the mutational status, expression, and/or function of a positive signal transduction factor (PSTF).
32. 32. A kit according to claim 31, wherein the means for testing a sample comprises a means for testing for the abundance or the mutational status of the NSTF and/or a means for testing for the abundance or the mutational status of the PSTF.
33. 33. A kit according to claim 32 for measuring the radiosensitivity of wild-type p53 cancer cells, which kit comprises a means for testing for the abundance of Raf-1 protein and a means for identifying wild-type p53 cells.
34. 34. A kit according to claim 32 for measuring the radiosensitivity of cancer cells, which kit comprises a means for testing for the abundance of p21 protein and a means for testing for the abundance of Raf-1 protein.
35. 35. A kit for measuring the resistance of p53 mutant cancer cells to the cytotoxic effects of chemotherapeutic agents, which kit comprises a means for testing for the abundance of cyclin D1 protein and a means for identifying p53 mutant cells.
36. 36. A kit according to claim 32 for measuring the resistance of cancer cells to the cytotoxic effects of chemotherapeutic agents, which kit comprises a means for testing for the abundance of p21 protein and a means for testing for the abundance of cyclin D1 protein.
37. 37. Use of a kit as defined in any of claims 31-36, to determine whether to treat a cancer by radiotherapy or chemotherapy.
38. 38. Use of a kit as defined in any of claims 31-36, to determine whether to treat a cancer with a first chemotherapeutic agent or with a second chemotherapeutic agent.