METHODS FOR CLINICAL DIAGNOSIS

Abstract: The present invention relates to methods for analysing chromosomes either for research or clinical diagnosis purposes, particularly for the diagnosis of chromosome abnormalities. The invention provides an improved method of increasing the proportion of cells in the metaphase stage of the cell cycle in order to facilitate the analysis of chromosomes. The method involves contacting the cells with a mitosis stimulating agent, for example, a protein phosphatase inhibitor such as okadaic acid (OA).

Description: Methods for Clinical Diagnosis Field of the invention The present invention relates to improved methods for analysing chromosomes either for research or clinical diagnosis purposes. Particularly, but not exclusively, the invention relates to materials and methods to increase the proportion of cells in the metaphase stage of the cell cycle in order to facilitate the analysis of chromosomes. Background of the invention For the accurate analysis of the majority of chromosomal abnormalities in cells, it is necessary to analyse cells in the metaphase stage of the cell cycle where the chromosomes are structurally condensed (Figure 1) . At this stage, the chromosomes can be stained with coloured dyes or can be subjected to hybridisation with fluorescent DNA probes in order to examine particular features of individual chromosomes, for example to look for deletions of translocations of particular chromosome segments. Through high resolution chromosome banding and the availability of a range of DNA probes, the analysis of chromosomes in relation to human disease has become commonplace. However, the metaphase stage of the cell cycle is transient and usually accounts for less than 2% of the cycling time of dividing mammalian cells. Thus, without any pretreatment , a typical preparation of mammalian cells will include very few cells in the metaphase stage and therefore will limit the level of chromosome analysis which is possible. For dividing cells, the use of mitosis-blocking agents such as colchicine, colcemid and vinblastine has proved invaluable for increasing the percentage of cells in mitosis (the "mitotic index") particularly in the chromosomal analysis of human peripheral blood lymphocytes. However, for many clinical samples, the cell cycling rate is very slow and few mitotic cells can be seen even with the use of mitosis-blocking agents. This is because the cell cycle has several biochemical "check-points" providing strict control on cell division and cycling. To accumulate enough cells in division that can be blocked in metaphase, in vitro culture of cells is commonly used. This causes an inevitable delay in the clinical diagnosis of chromosome abnormalities. Thus even human blood lymphocytes, specifically stimulated with phytohaemagglutinin (PHA) require 48-72 hours in vitro culture, amniotic fluid cells on average 16.7 days (Ntl. Qual. Ass. Scheme, Clin. Cytogenet, 1995) and skin fibroblasts several weeks. There is thus a need for methods to release such cells from their biochemical checkpoints in order to increase the metaphase index to enable effective clinical diagnostic analyses to be undertaken without delay and without the requirement for long-term in vitro culture. Spermatogenesis is the process during which diploid spermatogonia mature to haploid spermatozoa (Figure 3) . The majority of spermatogonia (type A) undergo an amplifying mitotic renewal pathway to maintain the required high number of stem cells. Only a minority (type B) start further differentiation through a premeiotic mitosis, which results in diploid primary spermatocytes . Such cells enter the first (reductional) division of meiosis, during which chromosome number reduces to half due to segregation of homologes to opposite poles at anaphase I. The resulting daughter cells subsequently undergo the second (equational) division of meiosis, which is mitotic-like, as chromatids are separated at anaphase II. These two divisions of meiosis produce four haploid round spermatids, which later mature to spermatozoa being differentiated biochemically and morphologically during spermiogenesis (Amelar, 1966; In: Infertility in Man: Diagnosis and treatment, pp2-30, F A Davis Company, Philadelphia) . The meiotic pathway takes days to be completed in contrast to the mitotic cell cycle, which can be measured in hours. For instance, prophase I of male meiosis takes about 40 days in the human, and the longest substage, pachytene, alone lasts around 16 days in the human. The second division, however, happens very quickly, and for that reason such spermatocytes are more rarely seen in the seminiferous epithelium. The whole process of formation and maturation of spermatozoa occupies about 74-76 days in the human male, and normally results in the production of around 200 million sperm cells every day. The meiotic cell pathway, in common with the mitotic cell cycle, consists of a specialised and highly regulated series of events, which are dependent upon the completion of each other. Thus the initiation of later events depends on the completion of earlier ones. This dependency is monitored by mechanisms, known as checkpoints. This regulatory system initiates each cell cycle transition only after receiving and processing a wide variety of growth- controlling signals, and also assures that the optimum condition is achieved in the cell. The eukaryotic cell cycle is controlled by periodic activation of members of a large family of cyclin dependent kinases (CDK) (Figure 4). CDKs are heterodimeric complexes, consisting of a protein kinase catalytic sub-unit, the monomeric CDK, and a cyclin activating sub-unit. CDK complexes catalyse the phosphorylation of certain protein substrates, crucial for each cell cycle phase (Busenberg and Tang 1994, J Math Biol 32,573-596) . Phosphorylation and dephosphorylation of proteins is one of the major regulatory cellular processes in eukaryotes, and its occurrence in an orderly manner is essential for normal cell division. Substrates or chemical compounds, which stimulate or inhibit the phosphorylation process during the cell cycle, would also affect the activity of CDK complexes and subsequently the cell division. Okadaic Acid (OA) is one of these compounds, which stimulates the phosphorylation of nuclear proteins in vi tro . OA is a polyether monocarboxylic fatty acid, which is isolated from the marine black sponge (Halicondria okadaic) . It is an inhibitor of protein phosphatase (PP), both PPl, an ATP-Mg2+-dependent phosphatase, and the related PP2A, a polycation-stimulated phosphatase (Bialojian and Takai, 1988, Bioche J 256,283-290). In rapidly dividing somatic cell lines OA has been reported to induce mitotic-like events. These include for instance, (1) premature chromosome condensation, disruption of nuclear lamina and formation of mitotic asters in the fibroblast cell line BHK21 of the Golden hamster (Yamashita et al, 1990, EMBO J 9,4331-4338), (2) mitotic arrest, chromosome over-condensation and chromatin degeneration in human leukemic cell lines (Zheng et al, 1991, J Biol Chem 266,10031-10034), (3) premature chromosome condensation at Gl/S transition in human myeloid leukemic cell lines (Ishida et al, 1992, J Cell Physiol 150,484-492) and HeLa cells (Ghosh et al, 1996, Exp Cell Res 227,165-169), and (4) mitotic arrest and depolymerization of spindles in the pig kidney cell line LLC-PK (Vandre and Wills, 1992, J Cell Sci, 1101,79-91). OA has also been reported to induce similar effects following microinjection of oocytes . Thus chromosome condensation has been seen in oocytes of Xenopus, starfish and mouse (Doree et al, 1989, J Cell Sci Suppl 12,39-51; Rime et al, 1990, Cell Differ Dev 29,47-58; Picard et al 1989, J Cell Bio 109,3347-3354; Alexandre et al, 1991, Development 112,971-980; Gavin et al, 1991, Exp Cell Res 192,75-81. The OA induction of meiotic maturation and germinal vesicle breakdown in oocytes of these systems have been shown to be associated with depolymerization of MI spindles (e.g. Picard et al, 1989, J Cell Biol 109,3347- 3354; Alexandre et al, 1991, Development 112,971-980), or with the presence of abnormal MI spindles (e.g. Gavin et al, 1991, Exp Cell Res 192,75-81). OA may also induce Mil arrest by affecting the spindle organisation in rat and mouse Mil oocytes; (De Pennart et al, 1993, Dev Biol 157,170-181) . Summary of the invention A recent study of isolated pachytene cells from male mouse has further shown that spermatocytes are sensitive to OA and mature to MI in vi tro after short term (2-6h) stimulation. The present inventors have applied a modification of this technique on crude human and mouse (for comparison purposes) testicular cell suspensions. It was the objective of this study to determine whether OA is effective on human spermatocytes, and if so, to what extent this rapid in vi tro maturation of prophase I to MI affects the chiasma frequency and distribution. In particular, they realised that it would be desirable to obtain an in vi tro stimulation system that could be used as an aid for the differential diagnosis of spermatogenic maturation arrest and changes in meiotic recombination/chiasmata, associated with male infertility. The present inventor's findings demonstrate that in addition to the rapid transition of meiotic pachytene cells to MI, OA stimulation also leads to a high frequency of spermatogonial metaphases and MII:s in both the human and the mouse. These surprising observations suggest that OA overrides the mammalian cell cycle checkpoints at all three phases of spermatogenesis, including premeiotic spermatogonial mitotic division, MI and Mil. The present invention is based on the discovery that the agent Okadaic Acid (OA) can cause a profound increase in the metaphase index of cells in crude extracts from testicular biopsies, in particular the mitotic spermatogonial cells preceding meiosis. Importantly, this effect is seen within 6 hours of in vitro stimulation. The invention also recognises, for the first time, that agents which override the biochemical check-points in the cell cycle in the same way as OA can be generally used in order to increase the mitotic index in clinical diagnostic analyses. Therefore, at its most general the present invention provides materials and methods relating to the preparation of cells for analysing chromosome using a mitosis stimulating agent. Preferably the agent is capable of driving interphase cells through part or all of the cell cycle phases to metaphase, for example, a protein phosphatase inhibitor such as OA or cell free extract from xeropus eggs in Ca 2+ modified medium. Such analysis may be important for the clinical diagnosis of cells for consitutional or acquired, structural and numerical abnormalities of chromosomes associated with for example, leukaemias and other haematological conditions as well as solid cancers. Further, it may be important following exposure to environmental agents (radiation and chemicals, known or suspected to cause chromosome damage) . In addition, chromosome analysis of metaphase chromosomes is important for a variety of other reasons, using cells from many different sources, for example, human, animal or plant origin. In a first aspect of the present invention there is provided a method for preparing a sample of cells for chromosome analysis comprising contacting an in vitro culture of cells with a mitosis stimulating agent such as a protein phosphatase inhibitor or an agent stimulating cell cycle progression, to increase the population of cells in metaphase. Preferably, the method further comprises the steps of bypassing gatekeeper proteins present on the surface of said cells prior to contacting with said mitosis stimulating agent for stimulating cell cycle progression. Where the gate keeper proteins are surface receptors for growth factors specific for each cell type, bypassing may be achieved by demembranating the cells (Banerjee S., et al (1994) Molec.Reprod.Dev.37:305-317) using for example, polyethylene glycol (PEG), lysolecithin or inactivated Sendai virus. One well known gatekeeper protein is retinoblastoma protein (Geradt et al (1994) J. Cancer 58(2)161-167). This is a major gatekeeper protein of the mitotic cell cycle for the transition of cells from Gl to S phase. Preferably this gatekeeper protein is bypassed either by the use of anti retinoblastoma protein antibodies which are commercially available or by the use of antisense RNA technology (Sala et al.; 1994: Cancer Research 54:1402-1406). The cells are then contacted with OA to activate mitosis/metaphase promoting factor. The method of the present invention has the surprising advantage of avoiding the need for in vi tro cell culture in order to analyse the cells. For this reason the method is ideally suited for analysis of, for example, 1) blood lymphocytes which usually take 48 to 72 hours of in vitro culture to accumulate enough metaphase cells; 2) bone marrow cells, a high mitotic index of which would provide a major advantage in the speed at which chromosome abnormalties leading to leukaemias and other related malignancies could be diagnosed; 3) amniotic fluid comprising fetal cells which usually require 7 to 28 days in culture for analysis. More rapid chromosome diagnosis will increase the speed in which abnormalities leading to, for example, Down Syndrome may be diagnosed; 4) skin fibroblasts which usually take several weeks in culture in order to diagnose; 5) solid cancers which are very difficult to grow in culture could be diagnosed more accurately and quicker if the metaphase index could be raised sooner. The method of the present invention therefore provides cells for clinical diagnosis including diagnosis for constitutional or aquired, structural and numerical abnormalities in chromosomes. This may involve a cell proliferation assay for diagnosing cells passing through GO, Gl S and G2 phase of the cell cycle. This diagnosis is particularly important for leukaemias and other haematological malignant conditions as well as for solid cancers. Further, it may be used to determine the malignancy grade of cells. Quite simply, the ease at which the potentially malignant cells pass through the GO, S, Gl and G2 phases, which are increasingly potentailly dangerous stages may be determined. At present there are no in vi tro tests that diagnose malignancy/cell prolifieration risk status. Current procedures include techniques such as cytology and histology which are not very accurate. As a further aspect, the present invention provides an in vi tro cell proliferation assay for determining the malignancy grade of a sample of cells, comprising the steps of contacting said cells with a protein phosphatase inhibitor and determining abnormalities in cells passing through GO, Gl, S and G2 phases of the cell cycle. This assay is particularly useful for leukaemias and solid cancer cells as described below. Cancer cells are those that have escaped from their normal DNA replication and cell cycle control. These cells can therefore proliferate indiscriminately. As long as increased cell proliferation is limited and localised to the original tumour site, it does relatively little harm. The problems arise primarily when cancer cells are able to proliferate substantially and to spread to other sites of the body. It is at this stage that they cause generalised cancer disease. Diagnosis of malignancy is usually performed by morphological analysis of cells in histological and cytological preparations. The present inventors has appreciated that there is an urgent need for improvements in the diagnosis of maligancy degree, in other words, to what extent tumour cells are able to escape from the usual cell cycle control, and the check points involved in this control. As cancer cells are more prone to proliferate, a higher proportion than normal of the cell nuclei are expected to be in the Gl, S, G2 and M phase of the cell cycle rather than in the "resting" GO phase. The cells at GO, Gl, S and G2 are all in the so called interphase of the cell cycle, which are very difficult or impossible to differentiate by traditional histological and cytological staining techniques. It is however possible, by immunocytochemistry and immunocytological techniques, using antibodies against key cell cycle proteins, to identify cell nuclei in their different phases of the interphase. Thus, for instance, cell nuclei at the late Gl phase are expected to show high amounts of the cdcδ protein (Piatti S., et al (1996)Genes and Development 10:1516-1531; and Hardy, C.F., et al (1997) PNAS USA 94:3151-3155); nuclei at the S phase are expected to show high amounts of Rb (retinoblastoma) protein; and cells at the G2 phase are expected to show predominantly increased levels of cdc25C protein (Lucibello F.C. (1995) The EMBO Journal 14(1): 132- 142) . In accordance with the present invention, OA may be used to cause a rapid transition of spermatological interphase cell nuclei to M phase and therefore provides a new in vi tro diagnostic device for the evaluation of cell proliferation potential and degree of malignancy. This Mitosis Acceleration Assay (MAA) is described in Figure 10. Such a MAA may be performed in accordance with the present invention by taking a tissue sample in the form of a biopsy from the area and carrying out the following steps. The biopsy could be from any cancer suspected area such as cells taken as a cervix smear, a bone marrow sample, when a leukaemia condition is suspected, or an aspiration or needle biopsy from a breast tumour. The cell nuclei, which are in the different phases of the cell cycle, may be identified by a test battery of antibodies against proteins abundant in respective Gl, S and G2 phases of the cell cycle. Part of the cell sample is exposed in cell culture to the mitosis stimulating agents (such as protein phosphatase inhibitors, e.g. OA, or cell free extracts from xenopus eggs in Ca 2+ modified medium (Morin et al., (1994) The EMBO Journal 13 (18)4343-4352; Lohka, M.J., et al (1983) Science 220,719-721; and Newport, J. , et al (1987) Cell 48,219- 230) , or OA following inactivation of the Rb protein) to determine how prone these nuclei are pass into M phase in vitro. Cell preparations are made both for diagnostic cancer chromosome analysis as routinely carried out in cytogenetic laboratories, and for immunocytology . To identify what proportion of cell nuclei are still in the GO in relation to those at Gl, G2 or M, following in vitro mitosis stimulation with the respective agents. The metaphase index together with the GO, Gl, S, G2 index may be used as prognostic indicators of malignancy degree and in vivo proliferation potential and thus prognosis. The present inventor further suggests that the MAA as described herein may be used to check the degree to which malignant cell nuclei are sensitive to in vi tro inhibition in proliferation, following inhibitory agents such as chemotherapeutic chemicals. For spermatogenesis cells, the present invention provides clinical analysis for determining the spermatogenesis maturation index. In all the above aspects the protein phosphatase inhibitor is preferably OA. The remaining text concentrates on the use of OA but the skilled person will appreciate that other similar agents as described above may be substituted. Somatic cells OA has previously been utilised as a means of understanding more about the mitotic cell cycle control in experimental systems. Fig 2 illustrates the cell cycle. After Metaphase (M) follows a Gap (Gl) period, then DNA synthesis (S) and then again a Gap (G2) . It has been noticed that OA added to artificial cell systems such as continuously in vitro growing cell lines can cause some chromatin condensation. There is no clear indication in these publications that the OA induced chromatin condensation would allow clear differentiation of chromosomes of clinical diagnostic value. Thus the chromosomes, although condensed to some degree, are generally described as "mitotic-like" rather than mitotic. Such chromosomes morphology is not good enough for clinical diagnostic purposes. The potential use of OA as an aid to current methodology for clinical cytogenetic diagnosis of chromosome abnormalities has to the inventor's knowledge not been considered. Neither is there any indication in the literature of such use in general analysis of metaphase chromosomes from any source be it human, animal or plant material . Germ line cells OA has also been used to study the specific meiotic cell cycle control in the germ line. It has been demonstrated that in experimental systems OA induces meiotic maturation with G2/M transition of oocytes of several species including the mouse. More recently it has also been demonstrated that OA can induce premature G2/M transition in cultures of isolated meiotic mouse testis cells, i.e. pachytene spermatocytes (Wiltshire et al, Dev. Biol. 169,557-567, 1995). These cells are in the first meiotic prophase stage. DNA replication has already occurred. OA treatment appears to abrogate cell-cycle check points that maintain the normally lengthy and complex processes of first meiosis. The specific processes taking place here include the eiosis-specific synapsis of the maternal and paternal homologous chromosomes and reciprocal recombination/crossing-over/chiasma formation between them. At first meiotic metaphase the paternal and maternal homologous chromosomes are held together by the chiasmata. At least one chiasma is thought to be obligate for the regular anaphase segregation. At first meiotic anaphase a reduction of the chromosome number takes place. The daughter cells will each normally contain half the original chromosome number. At the second meiotic division, the chromosomes split in a process similar to somatic mitosis. None of the publications dealing with the OA-induced chromatin condensation of either oocytes or spermatocytes have implicated that OA may carry the potential for use in clinical diagnosis of constitutional or aquired somatic chromosome aberrations as currently routinely performed by analysis of in vitro cultured blood lymphocytes, skin fibroblasts or amniotic fluid cells. The present inventor has found that short term exposure of OA (6 hours) may induce a dramatic effect on the, premeiotic, spermatogonial mitotic index with adequate chromatin condensation in crude cell suspensions from mouse and human testicular biopsy samples. The chromosome morphology achieved is of high enough quality to allow clinical diagnosis if extrapolated to samples used in routine clinical practice such as blood lymphocytes, skin fibroblasts and amniotic fluid cells. From previous experience their results with respect to the increased mitotic index of the spermatogonial cells in crude testicular suspensions, are unexpected. In addition the present inventor has found a dramatic increase not only in cells at Metaphase I (as shown previously) but also Metaphase II in testicular cell suspensions. It also appears that OA may induce rapid maturation of testicular spermatozoa, leading to increased motility. The present inventor has furthermore shown that OA may induce maturation of Bone Marrow Cells from interphase/prophase to metaphase . The effect of OA on cell cycle and clinical implications Completion of the cell cycle is dependent on the coordination of many different and complex processes. It is generally accepted that a series of changes in the cyclin-dependent kinases (CDKs) are involved. The inactive forms of CDKs are primarily comprised of two proteins, namely a kinase and a cyclin. Other proteins, the function of which are yet poorly understood may also be involved. The complexes undergo changes in their kinase and cyclin components, which are thought to derive the cell from one stage of the cell cycle to another. The particular cell cycle stage is determined by the respective proteins, which are activated or inactivated by phosphorylation, resulting from the activity of CDKs during that stage. Okadaic Acid (OA) is a polyether monocarboxylic acid known to be a specific inhibitor of protein phosphatases . Okadaic Acid can inhibit both type-2A and type -1 phosphatases, thereby activating the inactive Maturation-Promoting-Factor MPF to active MPF via its action on cdc25C. The present inventor has realised that some of the regulators of the premeiotic spermatogonial mitotic cells in the mouse and human male germ line are functioning in the same way as in the mitotic cell cycles of the soma. Therefore, the present inventor suggests that these observations with regard to the OA effect on testicular cell populations could form the basis for a new approach for the clinical diagnosis of chromosome abnormalities, using somatic cells such as blood lymphocytes, skin fibroblasts and amniotic fluid cells. This approach would obviate long-term cell culture. It will be apparent to the skilled person that the stimulation to maturation of not only spermatogonial cells but also both first and second meiotic metaphases may be used as a diagnostic aid in the assessment of reasons for reduced fertility due to spermatogenic maturation arrest. The present inventor has shown that the OA-stimulated cells demonstrate a normal recombination pattern, as measured by the chiasma frequency distribution at Metaphase I. The implication is that an OA assay could be used for the improved diagnosis of, for example, meiotic recombination mutants, thought to be associated with spermatogenic maturation arrests. The present inventor has also found that OA may increase the sperm mobility, which may be used for improvement with respect to fertility treatment . It will be apparent to those skilled in the art that Okadaic Acid is one of several possible mitosis stimulating agents which may be used to release cells from their cell cycle control check points. It is therefore possible that other agents may be used in the same way as outlined with respect to OA for the clinical diagnosis of chromosome abnormalities and analysis of metaphase chromosomes in general as well as the assessment and possibly also treatment of reduced fertility. Also included within the scope of the present invention are kits for preparing cells for clinical analysis comprising one or more or the following. 1) a mitosis stimulating agent (e.g protein phosphatase inhibitor such as OA, cell free extracts from Xenopus eggs in Ca 2+ modified medium, OA following inactivation of Rb protein, or retinoblastoma protein antibodies) ; 2) a demembranating agent such as lysolecithium or polyethyleneglycol (PEG); 3) antibodies with specific activities to be used as cytological markers for the nuclei in the Gl, S and G2 phases of the cell cycle; and/or 4) cell culture media, specific buffers and other agents for the making of chromosome preparations for cytogenetic analysis of metaphase chromosomes. Brief description of the drawings Modifications and further aspects and embodiments of the invention will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference. Experimental basis and embodiments of the present invention will now be described in more detail, by way of example only, with reference to the accompanying figures, in which Figure 1 shows mitosis chromosome condensation and the differences between Interphase, Prophase and Metaphase; Figure 2 illustrates the cell cycle. After Metaphase (M) follows a Gap (Gl) period, then DNA synthesis (S) and then a Gap (G2) ; Figure 3 shows the process of spermatogenesis during which diploid spermatogonia mature to haploid spermatozoa; Figure 4 shows, diagramatically, the eukaryotic cell cycle being controlled by periodic activation of members of a large family of cyclin dependent kinases (CDK) . CDK complexes catalyse the phosphorylation of certain protein substrates, crucial for each cell cycle phase; Figures 5 to 8 reflect the results shown in Tables 1 to 4; Figure 9 shows testicular cells prepared from three suspensions (unstimulated, OA stimulated and control (cultured without OA) ; and Figure 10 shows the steps involved in a mitosis accelerating Assay (MAA) . In A the steps include; 1) Make cytological preparations directly from biopsy material; 2) Expose preparations to cell cycle specific markers, using specific fluorescently labelled antibodies, and identify by conventional immuno fluorescence technology; 3) Evaluate cell cycle phase on a sample of individual nuclei, and calculate Cell Cycle Index, ie relative numbers of nuclei in G1,S, and G2. In B the steps include; 1) Make in addition cell suspensions from biopsy material; 2) Demembranate cells by Lysolecithin or alternative; 3) Add Mitosis Stimulating Compound, alternatively OA,Rb antibodies followed by OA, or cell free extract of Xenopus eggs in calcium modified medium; 4) Make cytological preparations from the respective final cell suspensions obtained by step Bl-3, evaluate Cell Cycle Index, using specific antibody staining as in step A; 5) Make traditional chromosome preparations as per routine in Cytogenetic Laboratories for diagnosis by analysis of metaphase chromosomes, following traditional staining technology, and in addition calcualte Metaphase Index. Detailed description Example 1 - Short-term in vitro OA Stimulation of Premeiotic Spermatogonial, First and Second Meiotic Metaphases in Human and Mouse Crude Testicular Suspensions Material and Methods Human testicular biopsy material (around 2mm 3 ) was obtained from two patients undergoing reverse vasectomy. Mouse testicular material of the strain Balb/C was obtained from the animal house at the University of Birmingham. One third of the testicular material was immediately cut using springbow scissors and placed in 1% TriSodium Citrate. Cells were teased out from the tubular material using a pair of curved forceps. Air-dried preparations were made according to Hulten et al. In: Human Cytogenetics, A Practical Approach, Eds.Rooney DE, Czepuldouski BH, IL Press, ppl93-221, 1992, or Evans et al. , Cytogenetics 3_3, 289-294, 1964, for human or mouse testicular material, respectively. The remaining two thirds of the material was placed in a C0 2 independent medium (GIBCOBRL) and macerated cells from the testicular tubules were divided into two equal aliquots. One aliquot was cultured with a 2μM final concentration of OA (Sigma) and the other without OA. Both aliquots were incubated at 35°C (human) or 37°C (mouse) for 6hr after which conventional air-dried preparations were performed as described. Cells were stained with Leishman stain and screened under bright field microscopy. The position of each metaphase cell was recorded using an England finder (Graticules, London) . Images of the cells were captured and stored on an Power Macintosh 8100/80 system utilizing Smartcapture software (Digital scientific) . These images were used later for scoring chiasmata and chromosome configurations. The frequency of cells in different stages of spermatogenesis was recorded in terms of the percentage of the cells from the total of the four cell types a) spermatogonial metaphase (SpM),b) prophase I including leptotene, zygotene, and pachytene (PI),c) metaphase I including diakinesis and metaphase I (MI), and d) metaphase II (Mil) (see Figure 9) . Fluorescence in si tu hybridization (FISH) was conducted according to Barlow and Hulten, Chromosome Research 4., 562-573, 1996, with some modifications. Immersion oil was removed from slides with 10 min in 4xSSC Tween 20. Then slides were placed twice in 3:1, (MetOH: Acetic Acid) each for 10 min to remove the Leishman stain. Three heterochromatic DNA probes were used, pUC 1,77 (lqh),pMR 9A (9qh), and D15Z1, to identify chromosomes 1,9, and 15, respectively. Probes were labelled with digoxigenin-11-dUTP or Biotin-11-dUTP (Boehringer Mannheim) , using a nick translation kit (Boehringer Mannheim) according to the manufacturer's instructions. Slides were screened using a Zeiss Axioskop epifluorescence microscope fitted with a Pinkel number 1 filter set and a cooled charged coupled device (Photometries) . Images of hybridized cells were captured electronically (as described above) for the comparison and identification of chromosomes in Leishman stained cells. For estimating the frequency of cells in the different stages of spermatogenesis, cells were classified into four categories; i) spermatogonial metaphase (SpM) ; ii) prophase-I (PI), including leptotene, zygotene, pachytene, and diplotene; iii) Metaphase-I (MI), including diakinesis and metaphase-I, and iv) Metaphase-II (Mil) . The index for each category was calculated in terms of the percentage of the cells from the total of all categories, i.e. other cell types were ignored. Results 1) General description Chromosome morphology, configuration and chiasma frequency were studied in air dried preparations of germ line cells after 6hrs incubation of crude testicular cell suspensions with and without OA. The most striking observation was the variety of cell types in OA-stimulated cultures compared to cultured cells without OA, which mostly contained arrested prophase I cells. The majority of cells in OA-stimulated cultures revealed a premature chromosome condensation, and the metaphase index was dramatically increased. A detailed study of chromosomes at spermatogonial metaphase, meiotic Metaphase I and II showed no differences in their morphology nor in their degree of chromatin condensation between OA-stimulated and unstimulated cells. 2) Cell type frequencies The Short term in vitro culture (6 hours) of crude testicular cell suspensions with OA at a final concentration of 2μM led to a dramatic increase in the number of prometa/metaphase cells compared to uncultured material. This effect was observed for both human and mouse crude testicular cell suspensions (Tables 1 and 2 and Figures 5 and 6 respectively) . The average number of cells at the first meiotic MI stage was increased dramatically in OA stimulated cells from 2.8 to 57% and from 4.2 to 64.9% in human and mouse, respectively. Interestingly, the frequency of the second meiotic, Mil cells was also increased by OA stimulation, nearly 6 fold in human and 3.3. fold in mouse. The most surprising and unexpected observation was the effect of OA on the frequency of pre-mitosis. Thus the crude testicular cellular material stimulated in vi tro by OA showed a large increase in the number of spermatogonial metaphases (SpM) . The frequency of this type of cells increased from 1.8 to 12.3% and from 3.4 to 12.3% in human and mouse testicular material, respectively. In contrast, the frequency of the meiotic prophase cells (PI) was dramatically reduced following in vi tro stimulation of cells with OA. Comparison of testicular cell suspensions cultured with OA and freshly prepared testicular cells revealed a 6 fold decrease in number of prophase I cells for human and a 7.2 fold decrease for mouse. Tables 1 and 2 and Figures 5 and 6 illustrate the above observations. An overall comparison is made between three different cell suspensions in terms of the cell type and frequency for man and mouse, respectively. The large rise in the frequency of metaphases in OA-stimulated cultures is evident in both systems. In contrast, cells cultured without OA for six hours showed no variation in cell types, and almost all cells appeared to be arrested at meiotic prophase (PI) . The majority of these cells were at the pachytene stage, and no other stages of meiosis or pre meiotic mitosis were observed. Table 1 : - Cell indices for human testicular cells SpM P MI Mil No Cells Male 1 1.30 94.00 1.70 3.00 300 Uncultured Male 2 2.40 91.60 3.90 2.10 333 Average 1.85 92.80 2.80 2.55 633 Male 1 13.30 17.30 52.00 17.30 150 OA culture " Male 2 11.30 13.70 61.90 13.10 388 Average 12.30 15.50 56.95 15.20 538 Male 1 0.00 99.50 0.50 0.00 200 Control Male 2 2.50 97.30 0.30 0.00 400 culture* Average 1.25 98.40 0.40 0.00 600 Data presented in percentage of the total Cells immediately prepared from fresh biopsy Cells prepared after 6 hr culture in medium with 2μM OA * = Cells prepared after 6 hr culture in medium without OA Table 2 : - Cell indices for mouse testicular cells SpM P MI Mil No Cells Male 1 3.75 90.50 3.50 2.25 400 Uncultured Male 2 3.00 88.00 5.00 4.00 200 Average 3.38 89.25 4.25 3.13 600 Male 1 12.97 13.51 62.16 11.35 185 OA culture Male 2 11.64 11.11 67.72 9.52 189 Average 12.31 12.31 64.94 10.44 374 Male 1 0.00 100.00 0.00 0.00 100 Control Male 2 1.00 96.00 0.30 0.00 100 culture* Average 0.50 98.00 0.15 0.00 200 Data presented in percentage of the total = Cells immediately prepared from fresh biopsy Cells prepared after 6 hr culture in medium with 2μM OA * = Cells prepared after 6 hr culture in medium without OA 3) Chiasma Frequency and Distribution The effect of OA after 6hrs in vi tro culture of testicular cell suspension on the formation of chiasmata at metaphase I was studied in comparison with uncultured cells. It was revealed that short term culture of cells with OA has no effect on the frequency distribution of chiasmata in either the human or the mouse. Table 3 shows the mean chiasma frequency of OA- stimulated and unstimulated human testicular material. There was no significant variation between the 2 individuals in terms of mean chiasma frequency per cell, indicating that in vi tro stimulation of cells with OA did not significantly affect the mean chiasma frequency. The mean chiasma frequency per cell in uncultured material was 48.3 and in OA stimulated cells 48.4. Chromosome 1,9 and 15 were identified by FISH. This provides the possibility to investigate the chiasma frequency per specific chromosome and thus obtain a more detailed picture of the effect of OA on crossing-over/chiasma frequency distribution. The mean chiasma frequency per human chromosome 1,9 and 15 in cultured testicular cells with OA was 3.9, 2.6 and 1.8, respectively, and did not differ from that of uncultured material, which was 3.8, 2.6 and 1.7, respectively. The effect of OA on chiasma frequency and distribution is presented in the form of a histogram in Figure 7 for human testicular cells. There is no clear difference between the average mean chiasma frequency per cell nor per selected bivalents. Table 3 : - Chiasma frequency and distribution of human males metaphase-I cells , uncultured and cultured with OA for 6 hours Individual Treatment Autosome No Cell Mean SD Range All 57 48.35 3.15 43=60 Uncultured Chr 1 61 3.72 0.7 2=5 Chr 9 43 2.53 0.5 2=3 Male 1 Chrl5 22 1.82 0.39 1 =2 All 50 48 3.24 43 =57 OA culture Chr 1 62 3.81 0.62 3 =5 Chr 9 62 2.59 0.56 2=4 Chr 15 27 1.93 0.47 1 =3 All 38 48.32 3.79 41 =57 Uncultured Chr 1 38 3.84 0.68 3 =5 Chr 9 38 2.61 0.5 2=3 Male 2 Chrl5 5 1.6 0.55 1 =2 All 101 48.71 3.66 42=60 OA culture Chr 1 114 3.98 0.59 3=5 Chr 9 116 2.53 0.5 2=3 Chr 15 110 1.69 0.48 1 =3 Table 4 illustrates the frequency of chiasmata per cell and the frequency of bivalents with the different numbers of chiasmata in OA-stimulated cells and unstimulated mouse testicular material. A similar pattern of chiasma frequency to that of the human was observed. Thus, the mean frequency of chiasmata per cell was not affected by the OA stimulation. Cells cultured with OA showed an average of 23.3 chiasmata compared to uncultured, having an average of 23.6 chiasmata. All autosomes formed bivalents with mainly one or two chiasmata. A few cells were observed which contained bivalents with three chiasmata. These cells were present in both OA-stimulated and unstimulated cells. Figure 8 illustrates the chiasma frequency and distribution for mouse. There is no significant difference in the number of chiasmata per cell. Bivalents with 1,2 or 3 chiasmata also showed similar frequency in both conditions. Table 4:- Chiasma frequency and distribution of male mouse metaphase-I cells, uncultured and cultured with OA for 6 hours Individual Treatment Chromosomes No Cell Mean SD Range All autosomes 43 23.19 1.31 21 =27 Uncultured Bivalent with 1 Chiasma 43 16.81 1.31 13 = 19 Bivalent with 2 Chiasmata 43 3.19 1.31 1 =7 Male 1 Bivalent with 3 Chiasmata 43 0.00 0.00 0 All autosomes 49 23.73 2.00 20=28 OA culture Bivalent with 1 Chiasma 49 16.22 2.00 12 = 19 Bivalent with 2 Chiasmata 49 3.76 2.00 0=8 Bivalent with 3 Chiasmata 49 0.00 0.00 0 All autosomes 76 23.32 1.29 22=26 Uncultured Bivalent with 1 Chiasma 76 16.68 1.24 14 = 19 Bivalent with 2 Chiasmata 76 3.28 1.21 0=6 Male 2 Bivalent with 3 Chiasmata 76 0.03 0.16 0= 1 All autosomes 80 23.41 1.08 22=28 OA culture Bivalent with 1 Chiasma 80 16.61 0.99 14 = 18 Bivalent with 2 Chiasmata 80 3.36 0.94 2=6 Bivalent with 3 Chiasmata 80 0.02 0.22 0=2 According to the above observations it would be reasonable to conclude that stimulation of cells with OA and the rapid transition of cells to metaphase I does not affect the frequency nor the distribution of chiasmata. Example 2 - Human Bone Marrow experimentation Samples of bone marrow cells from two patients with leukaemic conditions were suspended in Ham's F10 medium, and OA added to a final concentration of 2μM. These patients have had bone marrow transplants with bone marrow from a donor of the opposite sex. OA cell suspension and controls were incubated for 16 hours at 37°C. Preparations were made as per routine for cytogenetic analysis including 20 minute hypotonic pretreatment . The mitotic index was assessed from OA treated and control cell cultures. The majority of cells from OA cultures were in pro-metaphase or metaphase, very few cells were seen in these stages in untreated cell cultures. Fluorescence in-si tu hybridization technology was used to determine the sex chromosome constitution of the bone marrow cells which proved that they were the descendants of donor cells. As far as we are aware OA has not been successfully demonstrated previously to induce progression of normal (non cell-line) interphase/early prophase stages of human somatic cells to metaphase. Example 3- Production of Metaphase Chromosomes with MPF. METHODOLOGY: The aim of the present invnentor was to identify the most effective and efficient way of driving cells through Gl-phase of the cell cycle, as rapidly as possible, through the S- phase into mitosis (M-phase) . Having achieved this, the task of producing cells in metaphase devoid of all the prolonged and often demanding protocol of cell culture would have been achieved. In order to achieve this, the present inventor had to develop stategies that would enable her to break through the main two checkpoints that interfer with the rapid progression of cell into mitosis. Two locations in this regard are essential and important, namely: (1) The checkpoint at Gl/S-phase and (2) the checkpoint at G2/M-phase. Present scenarios at the check points . Giys-check point . Specific biochemical molecules determine the success of passage of cells from Gl into S-phase of the cell cycle. Work performed before the present invention had identified Cdk2-cyclin E and Cdk4/6-cylin D complexes as key performers in driving cells through the Gl/S checkpoint (Zavits, H. T., et al (1997) Curr. Opin. Cell Biol. 9: 773-781.). The activities of these proteins are independent of the role of Dap and its homolog CIP/KIP, molecules whose role of impeding the passage of cells through the checkpoint has been documented (Lane, M. E., et al (1996) Cell 87: 1225-1235.; De Nooij , J. C, et al,(1996) Cell 87: 1237-1247. ) . Cdk4/6-cyclin D and Cdk2-cyclin E phosphorylate members of the Retinoblastoma (Rb) family of "pocket proteins" (pRb, pl07 and pl30) . These phosphorylated proteins release the E2F family of transcription factors (E2F1-5) which activate the transcription of genes whose products are necessary for S-phase entery. Cdk2- cyclin E is also thought to phosphorylate other key substrates required to initiate DNA synthesis (Zavits, H. T., et al (1997) Curr. Opin. Cell Biol. 9: 773-781.). Overexpression of any of the Rb group of proteins (pRb, pl07 and pl30) can arrest cells in Gl phase (Zhu, L., et al (1993) Genes Dev. 7: 1111-1125.; Claudio P. P., et al(1994) Cancer Res 54: 5556-5560.) by binding and inhibiting the activities E2F family of transcription factors (E"F1-E2F5) thereby interferring with the transcription of genes necessary for entery of cells into S-phase (Bartek, J. , et al (1996) Curr Opin Cell Biol 8: 805-814) . Recent studies have also identified cul -1 , another class of negative regulators of Gl/S-phase checkpoint during development (Kipreos, E. T., et al(1996) Cell 85:829-839) . Stra tegy (Giys-phase checkpoint) : The main focus would be to interfere directly with the Retinoblastoma proteins as there are commercially available monoclonal antibodies against the protein (Geradts, J. , et al (1994) International Journal of Cancer. 58 (2) 161-167). Another alternative method would be to interfer with the activities of the precursor molecules such as the Cdk- cyclin complexes that phosphorylate and activate the Rb group of proteins. The functions of these precusor molecules can be abrogated by binding then to CIP/KIP and INK4 families of CKIs.

Claims: CLAIMS : 1. A method for preparing a sample of cells for chromosome analysis comprising contacting an in vitro culture of cells with a mitosis stimulating agent to increase the population of cells in metaphase. 2. A method according to claim 1 further comprising the steps of bypassing cell surface receptors present on the surface of said cells prior to contacting with said mitosis stimulating agent. 3. A method according to claim 2 wherein the cell surface receptors are bypassed by demembranation of said cells. 4. A method according to any one of claims 1 to 3 further comprising the step of inactivating a gatekeeper (checkpoint) protein present on said cell. 5. A method according to claim 4 wherein the gatekeeper protein is retinoblastoma protein. 6. A method according to claim 5 wherein said retinoblastoma protein is inactivated by a retinoblastoma specific antibody. 7. A method according to claim 4 or claim 5 wherein the gatekeeper protein is inactivated using RNA antisense technology. 8. A method according to any one of the preceding claims wherein the cells are blood lymphocytes, bone marrow cells, amniotic fluid containing fetal cells, skin fibroblasts, or solid cancer cells. 9. A method according to any one of the preceding claims wherein the chromosome analysis is for diagnosing constitutional or acquired, structural and numerical abnormalities in chromosomes. 10. A method according to any one of claims 1 to 8 wherein the chromosome analysis comprises a clinical cell proliferation assay for diagnosing cells passing through Gl, S and G2 phase to M the phase of the cell cycle. 11. A method according to any one of claims 1 to 8 wherein the cells are testicular spermatogenesis cells and the chromosome analysis is to determine spermatogenesis maturation index. 12. A method of analysing a sample of cells in vi tro for metaphase chromosome abnormalities comprising the steps of demembranating said cells to override surface receptors on the cell membrane; inactivating a gatekeeper (checkpoint) protein; activating mitosis/metaphase promoting factor by contacting said cells with a mitosis stimulating agent ; and analysing said chromosomes. 13. A method according to claim 12 wherein the gatekeeper (checkpoint) protein is retinoblastoma protein. 14. A method according to claim 12 or claim 14 wherein the gatekeeper (checkpoint) protein is inactivated by a specific antibody or by RNA antisense technology. 15. A method according to any one of the preceding claims wherein the mitosis stimulating agent is a protein phosphatase inhibitor capable of driving interphase cells through to metaphase . 16. A method according to claim 15 wherein the protein phosphatase inhibitor is okadaic acid (OA) . 17. A method according to any one of claims 1 to 14 wherein the mitosis stimulating factor is cell free extract of Xenopus eggs using Ca 2+ modified medium. 18. A method according to any one of the preceding claims when OA is used in combination with mitosis- blocking agents. 19. An in vi tro cell proliferation assay for determining the malignancy grade of a sample of cells, comprising the steps of contacting said cells with a mitosis stimulating agent and determining frequency of cells passing through part or all of the Gl, S, G2 and M phases of the cell cycle . 20. An in vi tro cell proliferation assay according to claim 19 further comprising the steps of demembranating said cells to bypass its cell surface receptors. 21. An assay according to claim 19 or 20 wherein the mitosis stimulating agent is OA. 22. An assay according to claim 19 or claim 20 wherein the mitosis stimulating factor is cell free extract of Xenopus eggs using Ca 2+ modified medium. 23. A kit for preparing a sample of cells according to any of claims 1 to 11 comprising materials for culturing cells and a mitosis stimulating agent. 24. A kit according to claim 23 further comprising materials for bypassing cell surface receptors and/or gatekeeper proteins. 25. A kit for carrying out an in vi tro cell proliferation assay according to any one of claims 19 to 21 comprising a mitosis stimulating agent.

Inventors: HULTEN MAJ, HULTEN, MAJ
Applicants: HULTEN MAJ, SIMEG LIMITED, HULTEN, MAJ
Application year: 1998
Main classification: G01N33
IPC codes: C12Q1, G01N33