Patent Application: US-51687707-A

Abstract:
the present invention relates to the diagnosis , prognosis or predisposition of developing cancer , especially lymphoma . the invention also relates to gene therapy and identification of potential new therapeutic agents for treating cancer such as lymphoma .

Description:
the present invention will now be further described by way of example and with reference to the figures which show : a : diagram of the targeting strategy for generation ndr1 −/− mice . schematic re - presentation of the ndr1 allele , targeting construct and targeted allele after re - combination . arrows indicate the positions of the pcr primers used to genotype mice . b : southern blot of kpni - digested dna showing successful recombination . binding site of the southern probe indicated in a . d : validation of the knockout by western blot . in thymus samples of ndr1 −/− mice no ndr1 kinase could be detected ; fig2 shows the up - regulation of ndr 2 upon ndr1 loss in various mouse tissues : a : expression pattern of ndr1 and ndr2 in mice as determined by qpcr ( from stegert et al . jbc 2004 ) b : ndr2 up - regulation in a variety of tissues from ndr1 −/− mice . tissues showing high expression of ndr1 in wild - type mice show a strong increase in ndr2 level . increase is weak in tissues showing no expression of ndr1 . fig3 shows increased lymphoma rate in aged ndr1 −/− mice , a : rate of high grade lymphoma in aged mice . out of 20 analyzed ndr1 −/− animals 11 ( 55 %) show lymphoma in various tissues . b : graphical representation of a . in addition the sex - specific rates of lymphoma incidents are included . c : tumor spectrum in aged ndr1 −/− and ndr1 +/+ mice . the spectrum is not altered upon ndr1 deficiency . fig4 shows immune - phenotyping and characterization of a representative lymphoma found in ndr1 deficient mice : a : h & amp ; e staining of kidney tissue invaded by tumor cells ( upper part ; 10 ×) b : staining of the same tumor with pax - 5 , a pan b - cell marker , the staining of the tumor cells is negative ( lower part ; 10 ×) c : staining with cd3 , a pan t - cell marker , tumor cells are positive ( right ; 10 ×) to investigate the biological function of murine ndr1 , ndr1 deficient mice were generated by targeting exon 4 of the ndr1 gene , as shown in fig1 a . the targeted disruption of the ndr1 gene locus was confirmed by southern blotting and pcr using tail - tissue samples from ndr1 wild type ( ndr1 +/+ ), heterozygous ( ndr1 +/− ) and mutant ( ndr1 −/− ) mice ( fig1 b and c ). on the protein level the absence of ndr1 was confirmed by using an antibody directed against the c - terminus of murine ndr1 . in thymus samples from knockout mice no signal for ndr1 could be detected ( fig1 d ). ndr1 knockout mice are viable , fertile and born in the expected mendelian ratio ( table 1 ). as younger ndr1 −/− mice show no apparent developmental defects , 14 - week - old mice ( 9 - 10 ) of each sex and genotype were subjected to a detailed analysis . included were organ weight measurements of brain , heart , kidneys , liver , ovaries , spleen , testes and thymus and histological examination of adrenals , bone marrow , brain , caecum , colon , duodenum , gallbladder , heart , ileum , jejunum , kidneys , knee joint , liver , lung , auxiliary and mesenteric lymph nodes , ovaries , pancreas , peripheral nerve , pituitary , rectum , spleen , sternum , skeletal muscle , skin , spinal cord , sternum , stomach , testes , thymus and thyroid with parathyroid and trachea ( data not shown ). the performed weight analysis of ndr1 −/− and wildtype animals revealed a slight ( but statistically not significant ) decrease in the bodyweight of 6 % in both sexes for the knockout animals . in addition a small weight decrease ( 14 %) of the testis of male knockout mice was recorded . however , there were no clear correlating microscopic findings . no other organs and tissues of ndr1 −/− mice showed any major weight or histopathological differences when compared to wildtype mice . there were also no significant differences between wild type and ndr1 deficient mice with regard to haematological evaluations , which included the analysis of complete blood cell counts as well as the analysis of hemoglobin and hematocrit values . the lack of an apparent phenotype in younger ndr1 −/− mice could be explained by a compensation of ndr1 function by the other isoform ndr2 . in tissue lysates from ndr1 ko mice a clear up - regulation of ndr2 was detected ( fig2 b ). this other isoform of ndr kinases shares 86 % amino acid identity and so far no differences in biochemical regulation and activation between these two kinases have been reported . interestingly , the observed increase in ndr2 levels seems to be tissue - specific as there is almost no change in the expression of ndr2 in tissues with low expression of ndr1 in wild - type . aged mice deficient for ndr1 show an increased rate of high - grade lymphoma as younger mice show no apparent phenotype upon ndr1 deficiency , we also analyzed aged mice ( 23 - 26 months ) to determine possible effects of ndr1 - loss on the development of age related lesions . in an initial study we used 20 knockout and 10 wildtype animals of both sexes . tissue sections of organs obtained from ndr1 −/− and ndr1 +/+ mice were stained with h & amp ; e and subjected to histological analysis , which identified several tumors as well as two cases of hepatis peliosis in the knockout mice . although the tumor spectrum is not altered in comparison to the wildtype , the incidence of lymphoma occurrence is strongly increased ( fig3 ). in 55 % of all ndr1 −/− animals high - grade lymphoma were identified , not only in lymphatic organs but also in other organs like liver , lung and kidney indicating a high rate of infiltration ( fig3 b ). interestingly , female mice seem to be more prone to develop high grade lymphoma . in 82 % of all female ko mice lymphoma were identified ( fig3 b ). we carried out immune phenotyping of the lesions and characterized them as being cd4 − cd8 + cd3 + high grade peripheral t - cell lymphoma ( fig4 ). interestingly the only observed lymphoma in wild - type mice was identified as being cd4 + cd8 − cd3 +, therefore a different kind of lesion ( data not shown ). this results clearly show an impact of ndr1 loss on the development of peripheral t - cell lymphoma . we found an up - regulation of ndr2 upon ndr1 ablation , interestingly suzuki et al . could show a potential link between ndr2 up - regulation and the development of b - cell lymphoma . future activities will address the question , whether ndr1 loss or ndr2 up - regulation is responsible for the observed increase in t - cell lymphoma rate . ndr protein kinases are highly conserved from yeast to men , indicating an important function of this kinases . however it seems rather surprising , that mice deficient for ndr1 show at least in young animals a rather mild phenotype . worms and flies only have one ndr kinase , sax1 and tricornered , and their ablation leads to more pronounced phenotypes . the organismal knockout of tricornered is lethal . due to an expansion of the kinome , mice and men have two ndr isoforms , therefore a functional compensation of ndr1 loss by ndr2 seems likely . indeed we could show , that ndr2 protein levels are increased in mice lacking ndr1 . aged mice , however , show a more pronounced phenotype with a strong increase in the rate of high grade lymphoma in ndr1 deficient mice . we observed in 55 % of analyzed ko mice the occurrence of cd4 − cd8 + cd3 + peripheral t - cell lymphoma , indicating a role of ndr1 in the control of normal t - cell function and regulation . an impact of ndr1 on the immunological roles of t - cells seems likely . 1 . manning , g ., whyte d . b ., martinez , e ., hunter t . & amp ; 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