DE10228187A1 - Test system for the detection of cancer - Google Patents

Abstract

The invention relates to a test system with means for detecting at least one anti-apoptotic member of the Bcl-2 family and with means for detecting at least one member of the Raf family, and uses of such a test system.

Description

Field of the Invention

The invention relates to a test system to detect cancer or the risk of cancer, Screening method for finding substances for treatment and / or Prophylaxis of cancer, cells and transgenic non-human mammals to carry out the screening process and uses by means of such screening Process of identified active substances.

Background of the Invention and state of the art.

The majority of tumors are socialized with genetic changes. These include in particular point mutations of the Raf-activating oncogenes, mainly of ras (Fong and Minna, Clin Chest Med 23: 83-101 (2002)) and overexpression by C-Raf (Rapp et al., J Int Assoc for the Study of Lung Cancer 4: 162-167, (1988)) or Bcl-2 (Ranger et al., Nat Genet 28: 113-118 (2001)).

Raf kinases are in the process of signal transmission of receptors of different growth factors in crucial Way involved.

On the one hand, this participation runs through the classic Raf-MEK-ERK signal transmission path, what cell growth, differentiation, proliferation and survival regulated (Daum et al., Trends Biochem Sci 19: 474-480 (1994)). A decisive factor here seems to be the intensity of the Raf signal to be: low intensity stimulates proliferation and high intensity inhibits cell division and differentiation (Kerkhoff and Rapp Oncogene 17: 1457-1462, (1998)). Three Raf isoenzymes (A, B and C Raf) are known to date. This have overlapping Functions in the control of the classic signal transmission path. However, they also each have specific functions in the Interaction with metabolic enzymes, proteasome regulator proteins and Bcl-2 family proteins. Raf kinases also affect the Raf-MEKK-IKK-NFkB signal transmission path. Activation of NFkB is associated with cell transformation by Raf-Oncogene. Dominant-negative mutants that activate of NFkB also inhibit cell transformation by oncogenes C-Raf (Baumann et al., PNAS USA 97: 4615-4620 (2000)).

Another signal transmission path connects C-Raf to Bcl-2, some Bcl-2 interacting proteins like BAG-1 and the pore protein VDAC (voltage-dependent anion channel) in the outer membrane of the mitochondria. C-Raf binds to these proteins, is with them on the outer membrane the mitochondria localizes and cooperates with Bcl-2 in inhibition cytochrome C release and apoptosis (Wang et al., Oncogene 9: 2751-2756 (1994); Wang et al., Cell 87: 629-638 (1996); Wiese et al., Nat Neurosi 4: 137-142 (2001); Troppmaier et al., Curr Top Microbiol Immunol 182: 453-460 (1992).

The inhibition of apoptosis by However, C-Raf is not necessarily linked to the presence of Bcl-2, since C-Raf localized on mitochondria also without Bcl-2 anti-apoptotic can act (Zhong et al., Oncogene 20: 137-142 (2001)).

Studies on the formation of tumors in mice dependent on resulted from the expression of Raf alone or in combination with myc, that the inclination or susceptibility to the emergence of Raf or Myc-expressing Tumors of a few, possibly is only controlled by one gene (Klinken et al., J Virol 63: 2411-2414 (1989)). This susceptibility gene was previously unknown. His knowledge would make it possible to identify patients which with an increased Probability of developing tumors. In such patients could Targeted prevention programs to prevent tumors or for their early detection be used.

The Bcl-2 protein family exists both from inhibitors and from promoters of apoptosis. Your essential Functions are that of an ion channel and a binding protein.

Inhibitors of apoptosis include Bcl-2, Bcl-X, Bcl-w, Brag-1, Mc-1, and Al1. Members of this group own 4 domains, inclusive the BH4 domain through which these inhibitors of apoptosis C-Raf and other regulatory proteins bind and couple to the mitochondrial membrane.

Promoters of apoptosis include Bax, BAD, Bak, Bcl-Xs, Bid, Bik, and Hrk. These promoters lack at least one domain in which most cases (with the exception of Bcl-Xs) the BH4 domain. (Overview see Kroemer et al., Nature Med, 3: 614 (1997); Chang et al., EMBO J 16: 968-977 (1997)).

All apoptosis inhibitors of the Bcl-2 family are considered oncogenic. Your expression will, for example induced by Myb, Ras, and AML-ETO (Klampfer et al., PNAS USA, 93: 14059-14064, (1996); Taylor et al., Genes Dev 10: 2732-2744, (1996)). The expression of Bcl-2 is common increased in myeloid leukemia and in tumors of the esophagus Stomach, colon and rectum and often inversely proportional expression of p53 and c-myc (Choi et al., Oncogene 11: 1693-1698, (1995); Delia et al., Blood 79: 1291-1298, (1992), Popescu et al., Eur J Cancer 34: 1268-1273, (1998); Torzewski et al., Clin Cancer Res 4: 577-583, (1998) Kroemer and Reed, Nat Med 6: 513-519 (2000)).

Bcl-2 apoptosis promoters Family are considered tumor suppressors. Negative mutations can occur in Tumors are found (Rampino et al., Science 275: 967-969, (1997), Ouyang et al., Clin Cancer Res 4: 1071-1074, (1998)).

All members of the Bcl-2 family form homodimers or selected ones Combinations of heterodimers. The formation of heterodimers from Inhibitors of apoptosis with promoters of apoptosis neutralize the antiapoptotic effect of the inhibitors.

In view of the oncogenic potential of Bcl-2 or Raf, attempts have been made to specifically inhibit the translation of bcl-2 by antisense oligonucleotides (Tamm et al., Lancet 358: 489-497 (2001); Ackermann et al., WO 00/40595 ; Zangemeister-Wittke et al., WO 00/66724 ; Warrel et al., WO 02/17852 ). Alternatively, small molecular inhibitors (Huang et al., WO 00/04901 ; Hockenberry et al., WO 01/14365 ; Wang et al., WO 02/13833 ) or antibodies against Bcl-2 (Rüben et al., WO 01/57060 ) used to inhibit Bcl-2.

Studies on the interaction between Bcl-2 and Raf with regard to cytokine-dependent proliferation of hematopoietic Cells revealed Bcl-2 overexpression the cytokine dependence overexpressing by Raf Could not reduce cells and, on the other hand, Bcl-2 overexpression a protective effect on Raf overexpressing Cells in that Bcl-2 exited from the S and G2 / M phases delayed cell division (Moye et al., Leukemia 14: 1060-1079 (2000)). Other systems are known to have high levels at Bcl-2 can inhibit cell cycle progression (Pietenpol et al., Cancer Res 54: 3714-3717 (1994)); O'Connor et al Curr Opin Cell Biol 12: 257-263 (2000)).

Technical problem of the Invention.

The invention is technical Underlying problem, the diagnostic certainty for cancer, in particular risks of cancer, improve, and means of to provide improved treatment for cancer.

Basics of the invention and preferred Embodiments.

To solve this technical problem the invention teaches a test system with means for detection at least an anti-apoptotic member of the Bcl-2 family and with funds for the detection of at least one member of the Raf family.

The invention further teaches the Use of a test system according to the invention for (in vitro) diagnosis of cancer or a risk cancer in human and / or non-human Mammals. A diagnostic method can thus be carried out with the invention, taking in vitro or in vivo cells from a patient for co-expression a member of the Bcl-2 family and a member of the Raf Family, possibly overexpression a member of one or both families.

As a further use of the test system according to the invention the invention teaches use in screening for substances which in a cell that is both an antiapoptotic member of Bcl-2 Family expressed as a member of the Raf family constitutively expressed reduced the expression or function of the member of the Bcl-2 family.

The invention further teaches one isolated human or non-human cell or a transgenic non-human Mammal, in particular Rodent, for use in a method according to the invention, wherein the cell or cells of a defined tissue of the transgenic non-human mammal both an anti-apoptotic member of the Bcl-2 family as well as a member of the Raf family and a screening process for active substances for the treatment of cancer, the cell or the non-human mammal a prospective active substance or a mixture of such substances can be supplied in a physiologically effective dose, after which the Quantity and / or activity of the anti-apoptotic member of the Bcl-2 family is measured, taking the amounts and / or activities of the antiapoptotic member the Bcl-2 family before and after the addition of the prospective active substance or the mixture can be compared with each other and being a prospective Active substance that has led to a reduction in the amount and / or activity, if necessary after deconvolution.

Finally, the invention teaches the use of an active substance identified by means of a method according to the invention for the production of a pharmaceutical composition for the treatment of cancer. The invention can be used to practice a method for the treatment of cancer, wherein a patient is given a physiologically effective dose of the identified active substance in a suitable galenical preparation, if necessary after prior use of the diagnostic method explained above. The administration can take place in a defined treatment plan, the dosage (amounts of a dose and time intervals between doses) with the proviso of a demonstrable reduction in the amount and / or activity of the member of the Bcl-2 family or a pathologically detectable improvement in the Clinical picture takes place.

The invention is based on the knowledge that that the expression of an antiapoptotic member of the Bcl-2 family's receptivity of a cell for tumor development by the Raf protein. It was found within the scope of the invention that, for example, the Expression of Bcl-2 accelerates tumor development by an oncogenic Raf protein, this effect of Bcl-2 is not due to constitutive expression, d. H. enhanced effect of the oncogenic Raf protein can be compensated. Evidence of, for example Bcl-2 and oncogenic Raf is ultimately predictive of tumor development in the tissue from which the cells of the finding originate.

Anti-apoptotic members of the Bcl-2 Family are for example Bcl-2, Bcl-X, Bcl-w, Bfl-1, Brag-1, Mcl-1 and A1. Bcl-2 is preferably detected in the context of the invention.

For example, members of the Raf family are C-Raf, A-Raf, B-Raf and oncogenic mutations of C-Raf, A-Raf or B-Raf. Preferred in Within the scope of the invention, C-Raf or mutated C-Raf is detected.

Evidence of expression at least a member of the Bcl-2 family and at least one member the Raf family is carried out by detecting the mRNA or via the Detection of the protein. Detection of the respective mRNA is preferred with the help of the reverse transcriptase - polychain reaction (RT-PCR) carried out with the help of specific primers, the detection of each Proteins preferably with the help of specific ligands, for example with the help of specific antibodies or mimicry compounds of such antibodies. Both detection methods are known to the person skilled in the art. antibody against Bcl-2, for example, are commercially available (Santa Cruz Biotech, Dako).

The constitutive expression in the frame a transgenic non-human mammal or an isolated one transformed human or non-human cell be set up in that the Bcl-2 family gene and Raf family gene under control suitable promoters is provided. In the case of the Raf gene comes for example the SP-C promoter in question. Then the constitutive takes place Expression predominantly in lung cells. In the case of the Bcl-2 gene comes for example the HSK promoter in question. This allows in particular screening for active substances which are used to treat lung cancer are suitable. For other indications can easily be other suitable promoters apply.

With the help of the test system according to the invention, this means through the detection of at least one anti-apoptotic Member of the Bcl-2 family and at least one member of the Raf Family in a cell or in a tissue, for example in bronchial epithelial cells, let yourself according to this Invention say whether there is a risk that the particular Will transform tissue into a tumor tissue. If this risk is detected, enables this test system the early Application of preventive measures, such as avoiding carcinogenic exposure Substances and the use of dietary, pharmacotherapeutic and / or surgical measures.

To the invention, preferably in suspension held cells tested with the test system according to the invention will be the one to be checked prospective active substance in concentrations of 0.1 nmol add up to 100 μmol and it will checked, whether the active substance is capable of expressing an antiapoptotic Member of the Bcl-2 family and / or a member of the Raf family to reduce.

Definitions.

The sequences of the genes mentioned or proteins are known from the NIH gene database (AccNrs .: Bcl-2: M14745; Bcl-XL: Z23115; Bcl-w: U59747; MCL-1: L08246; A1 / Bfl1: NM 004049; Diva / boo: NM 020396; NRH (human homologue of NR 13): CAD30221; BAD (containing the BH-3 domain): AF031523; C-Raf: X03484; A-Raf: X04790; B-Raf: M95712; the three the latter genes are members of the Raf family, the previous ones are anti-apoptotic members of the Bcl-2 family). in the However, homologs are also included within the scope of the invention, wherein homology with the known genes or proteins at least 80%, preferably more than 90%, most preferably more than 95%. In the case of nucleic acid sequences are also complementary or includes allelic variants. There are also sequences comprises which are only partial sequences of the known sequences with the proviso that these Partial sequences in the case of nucleic acids for hybridization with a sufficient nucleic acid according to the invention Length, at least 50 bases, and in the case of proteins or peptides with at least same affinity bind to a protein or peptide specific target molecule. Furthermore, all nucleic acids hybridizing with the known nucleic acids are included, namely those those under stringent conditions (5 ° C to 25 ° C below the melting temperature; see additional J.M. Sambrook et al., A laboratory manual, Cold Spring Harbor Laboratory Press (1989) and E.M. Southern, J Mol Biol, 98: 503ff (1975)) hybridize. In particular, point mutations of the known genes are also included, such as for example oncogenic C-Raf mutants. It is understood that the invention also includes expression cassettes, i. e. one or more of the used according to the invention nucleic acid sequences for a Bcl-2 member and Raf member, each with at least one control or regulatory sequence.

In connection with uses according to the invention, the terms include the Bcl-2 families and / or Raf families nucleic acids or proteins in addition to the full lengths of the sequences mentioned (see also the preceding paragraph) and partial sequences thereof, with a minimum length of 12 nucleotides, preferably 30 to 90 nucleotides, in the case of nucleic acids and a minimum length of 4 amino acids, preferably 10 to 30 amino acids, in the case of the peptides or proteins.

The terms of diagnosis and / or cancer treatment also includes detection and / or treatment of metastases from primary tumors.

Mimicry molecules are compounds that the emulate a variable region, in particular the binding region of an antibody and bind in the same place as a target molecule lying antibodies.

The term antibody includes polyclonal Antibody, monoclonal antibodies, non-human, human and humanized antibodies, as well as phage display antibodies, however also chimeric antibody and anti-idiotypic antibodies as well as specific fragments of the light and / or heavy chain of the variable range underlying antibodies above Art. The production or production of such antibodies with given immunogens is well known to the average person skilled in the art and does not need to be explained become.

One in the context of a detector substance established reporter group is an atom, molecule or compound which in conjunction with an assay placed thereon the reporter group and thus associated with the reporter group Allows connection or substance. examples for Reporter groups and associated detection methods are: 32P labeling and intensity measurement using phosphoimager. Many other examples are of ordinary skill in the art known and need not the detailed list.

As part of the above definition across from The narrow sense of the word also includes expanded definitions the certain terms in the narrow sense of the word.

EXAMPLES

Example 1: Production and typing transgenic mice.

The following mouse strains were published according to the Methods prepared as follows: mice, transgenic for a shortened activated C-Raf Kinase (C-Raf B × B) under the control of the surfactant protein-C (SP-C) promoter (Kerkhoff et al., Cell Growth Diff 11: 185-190 (2000)), mice, deficient in bcl-2 (Michaelidis et al., Neuron 17: 75-89 (1996) and mice, transgenic for humane bcl-2 under the control of the HSK promoter (Domen et al., Blood 91: 2272-2282 (1998)). Strains of mice were bred by hybridization and back-crossing express the respective transgenes as follows: SP-CcrafBXB / bcl-2 (- / -); SP-CcrafBXB / bcl-2 (+/-). Furthermore, the triple became by crossing transgenic mice SP-CcrafBXB / bcl-2 (- / -) / HSKbcl-2 generated.

The respective mouse strains were genotyped using the PCR (Invitrogen reagents) and primers, specific for the respective transgenes. The following primers were used in particular (see also the sequence listing Seq. ID 1 to 6 in the following order):
craf: 5'-ATC TCC GTG CCA TTT ACC-3 '
SP-C 5'-GAG AGG AGA GCA TAG CAC-3 '
bcl-2 sense: 5'-CAC CAG AAT CAA GTG TTC GGT G-3 '
bcl-2 antisense: 5'-GGT AGC GAC GAG ARG AGT CAT C-3 '
H2K sense: 5'-CGC GGA CGC TGG ATA TAA AGT C-3 '
H2k antisense 5'-ACA TCT CCC GCA TCC CAC TC-3 '

From mice transgenic for SP-C crafBxB known to have all adenomas with a relatively short latency of the lungs (Kerkhoff et al., Cell Groth Diff 11: 185-190 (2000)).

Example 2: Influencing the emergence of tumors.

mice deficient in bcl-2, live up to about a year without an accumulation of To have tumor diseases (Veis et al., Cell 75: 229-240 (1993)). mice which express human Bcl-2 also lived without a cluster tumors (Domen et al., Blood 91: 2272-2282 (1998)).

In mice, transgenic for SP-CcrafBXB The level of endogenous bcl-2 RNA and protein rose up to about 1.5 months and then remained stable at this elevated level. SP CcrafBXB transgenic animals lacking an allele of the bcl-2 did not show any Differences in the latency of tumor development, during animals, which lacked both alleles, a significant delay in the development of Had tumors. In the triple transgenic mice, the latency was the However, tumor development is again approximately the same as in bcl-2 (+ / +) mice.

To answer the question of whether Bcl-2 influenced the initiation or promotion of tumors The number of tumor sites per mm ² lung was examined histologically in 1.5, 2.5 and 6 month old mice. All transgenic mouse strains showed normal lung development macroscopically as well as histologically.

Transgenic in Bcl-2 negative SP-CcrafBXB mice However, after 1.5 months, there were no tumor foci, after 2.5 months significantly fewer tumor areas and the same amount only after 6 months Detect tumor centers as transgenic in Bcl-2 positive SP-CcrafBXB Mice. On the other hand, in the triple transgenic mice, which had an increased expression of human Bcl-2 exhibit the development of the tumor focus in comparison to SP-CcrafBXB transgenic mice not accelerated.

In lung sections from 6 months old mice were additional the number of apoptotic cells using the tunel assay (In situ cell detection POD kit, Roche) and the number in the S phase proliferating cells located in the lung tumors with the help of antibodies against PCNA (PharMingen). While in the number of apoptotic Cells showed no clear differences were found for Bcl-2 negative mouse strains one significantly reduced number of proliferating tumor cells in comparison to all other tested Strains of mice.

In summary, it can be said that the presence of Bcl-2 enhances Raf's oncogenic effects such that Tumors with shorter Latency occurs. This effect of Bcl-2 cannot be caused by constitutive expression can be compensated for example by C-Raf. The detection of Bcl-2 and oncogenic Raf in a tissue is consequently predictive of the development of a tumor in this tissue.

Example 3: Determination expression level of Bcl-2 and Raf in tissue biopsies semi-quantitative RT-PCR

In this example, the expression of Bcl-2 and C-Raf mRNA in biopsies of tumor tissue with non-degenerate Compared tissue. The biopsy material is obtained accordingly the location of each tumor, it becomes both healthy tissue as well as tumor tissue.

Starting from 0.5-30 mg Tissue (ideally 10 mg) depending on After the biopsy, the type of tumor is then processed mRNA isolated using a suitable method. In the present example from lung tumor tissue and neighboring normal lung tissue with the commercially available RNeasy kit from Quiagen according to the manufacturer's instructions isolated the mRNA.

The mRNA is then reverse transcribed, for example using the Quiagen Omniscript RT Kit in accordance with the manufacturer's instructions and an oligo-dT primer. A PCR reaction is now carried out with a cycler, for example the Robo-Cycler from Stratagene, with the following primers and conditions (Leng et al., Eur J Pharmacol 409 (2000): 123; Deshpande and Moore, Mol Cell Biochem 178 (1998 ): 47)): Primer (see also the sequence listing Seq. ID 7-12 in the following order):

The PCR is in a volume of 50 ul with 1 ul RT product, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 1.5 1.5 mM MgCl2, 0.3 µM of the corresponding specific 5 'and 3' primers and 2 U Taq DNA polymerase (Stratagene) performed. The conditions are like follows: 94 ° C, 5 min, 30 cycles of bcl-2 / b-actin or 35 cycles of c-raf-1 / b-actin at 94 ° C 45 s, 55 ° C 45 s and 72 ° C 1 min. Finally, there was a final extension at 72 ° C for 5 minutes carried out. The products are over Agarose gel electrophoresis separated and the relative product concentration is determined densiometrically. The quotient of the relative concentration of the bcl-2 or c-raf-1 product formed with the b-actin product, which is the relative Expression of bcl-2 or c-raf-1 is equated.

Then the relative expression of bcl-2 and c-raf-1 compared between tumor tissue and normal tissue. A clear increase was observed in the present example.

SEQUENCE LISTING

Claims (11)

Test system with means for detecting at least one anti-apoptotic Member of the Bcl-2 family and at least with means for detection a member of the Raf family. Test system according to claim 1, wherein the means for detection detector substances which are specific to are mRNA or protein of the respective member, the means in the case of mRNA substances for the specific amplification of the can include mRNA. Test system according to claim 1 or 2, wherein the anti-apoptotic Acting member of the Bcl-2 family is selected from the group consisting of from "Bcl-2, Bcl-X, Bcl-w, Bfl-1, Brag-1, Mcl-1 and A1 ". Test system one of the claims 1 to 3 with the member of the Raf family selected from the group consisting of C-Raf, B-Raf, A-Raf, mutated C-Raf, mutated A-Raf and mutated B-Raf ". Test system according to one of claims 1 to 4, wherein at least one of the detector substances is an antibody. Use of a test system according to one of claims 1 to 5 to diagnose cancer or a risk of cancer in human and / or non-human mammals. Use of a test system according to one of claims 1 to 5 for screening for substances that are in a cell that both an anti-apoptotic member of the Bcl-2 family as well as a member the Raf family expresses the expression or function of the member the Bcl-2 family. Transgenic non-human mammal for use in one A method according to claim 7, in particular Rodent, the cells of which one defined tissue both an anti-apoptotic member of Bcl-2 Family as well as a member of the Raf family. Isolated human or non-human cell for use in a method according to claim 7, which is both an anti-apoptotic Member of the Bcl-2 family as well as a member of the Raf family constitutively expressed. Process for screening for active substances for the treatment of Cancer, wherein cells according to claim 9 or a non-human mammal according to claim 8, a prospective active substance or a mixture of such Substances are added in a physiologically effective dose, followed by the Quantity and / or activity of the anti-apoptotic member of the Bcl-2 family is measured, taking the amounts and / or activities of the antiapoptotic member the Bcl-2 family before and after the addition of the prospective active substance or the mixture are compared with one another and a prospective active substance, possibly after deconvolution, which leads to a reduction in the amount and / or activity guided has been selected. Use of a by means of a method according to one of claims 7 or 10 identified active substance for the production of a pharmaceutical Composition for the treatment of cancer.