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Mitochondrial DNA deletions are common in basal and squamous cell carcinomas
ESDR I JSID I SID Abstracts
0289
0292
SPECIFIC DETECTION OF MELANOMA CELLS IN THE BLOOD BY ML4 RT-PCR I, w S ,
MI’IXKHONDRIALDNA DELETIONS ARE COMMON IN BASAL AND SQUAMOUS CELL CARCINOMAS. p w Depts. of Demmtol. and Molecular Medicine. Emory Univ., Atkmta GA, USA The purpose of thii study was to dotennine the frequency of mitochondrial chranawmal deletions in normal skin and in s(~“a”touscell (SC0 and basal cell !BCC) carcinomas. Mitocbontia represe”t the energy’powerho”se&fthe’ceI1but as a consequence produce a tremendous amount of reactive oxygen species (ROS). I” additkm to the i”“i”sically produced ROS, keratinocytes must deactivate ROS pmd”ced by expo.we to ulaaviokt (UV) radiation. Oxidative stress is induced when prooxidants exceed me capacity of me a”tioxida”t mecha”is”ls u) detoxify than. Pote”l!al long teml cansequenees of ox&rive siress include .&I damage, a&8. cell death and cancer. Mitochoodrial DNA (mrDNA) is a 16569 “uckotide double strmded circular genetic element located in the cytoplasm. mtDNA is more sensitive than nuclear DNA to the effects of UV induced oxidative damage. Mutations pmdwd in mtJJNA are not readily repaired and as a result deletions of various mxle&i& le”8th.soccw. Downstream effects of the mtDNA deletions include ineffiiient oxidative phosphorylation and inneased production of ROS. In this study we evaluated 12 samples of nomul skin surrounding skin canfrom predominately older individuals as well as 18 BCCs and 15 SCCs for the presence of mtDNA deletions using long extension PCR to mnplii the entire mitochondrial genome from harvested genomic DNA opposing primers were located in the.cytochrome b gene. Reaction products were electmpboresed on agamse gels and evaluated for deletions by size. The mitcchondria? ori@” of the K!R pmducu wa( confirmed by southern blotting. 66% of the nommi skm samples displayed mfDNA deletions as did > 90% of the SCCs. Interestingly only 33% of the BCCs showed mtDNA deletions. We conclude that mtDNA deletions a comma” in normal ski” in the region of skin cancers as well as in the BCCs and SC& We hypot!xsiLe that these deletion are induced in part by “lwviolet radiation and play a mechanistic role in skin callfer by increasing axidative stress.
0290
0293
EXPRESSION AND REGULATION OF THE h4!SMATCH-REPAIR-ENZYME HMSH2 IN HUMAN SKIN TUMORS. 1. Reichrath’. K. Rsss’. P. Go&vein’. C. Welte?. M. S&f&, K.D. Zanr’ and W. Tileen’. Departments of ‘Dermatology and ‘Human Genetics, “niverwty of the Saarland, Homburg, Germany. Mutations in the coding regions of methyl-directed nusmatch-repair genes, particularly LMSH-2, are involved in microsatellite instability and therefore seem to play a crucial role in hereditary nonpolyposis co!orecta! cancer. As we could previously demonstrate a phynmlogical connection between the W-inducible tumor-suppressor ~53 and LMSH-2 by exploring a site with homology to the ~53 consensus sequence in the promotor @on of hh&H-2, we now studied thekxpresslon and regulation &hMSH-2 in human skin tumors. We analyzed immunoh~sttochemically the expression of hMSH-2, ~53 and the proliferation marker Ki-67 on paraffin sections of normal human skin end different epithclial skin rumours [malignant melanoma, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), Bowen’s disease and actinic kemtoses]. Furthermorewe investigated the UVB-
Expression of hMSH-2 was upregulated in all malignant skin tomours, most markedly tn BCCs and Bowen’s disease, as compared to normal human skin. HMSH-2 up-ngulat~on was mostly pronouncedin turnoursrevealing strong p53 immunoreactivity Expression analysis by means of RT-PCR m UVB-treated SCL-I cells revealed an induction of p53 mRNA and subsequent up-regulation of hMSH-2 mRNA in vitro. Our findings indzate that LMSH-2 is strongly expressed in malignant skin tumors, most markedly in those which reveal strong pS3 immunoreacfivity. The functional expression-analysis pomts to the fact that LMSH-2 is directly mvolved in ~53 mediated DNA-repair following UVBdamage.
LOSS OF HETEROZYGOSITY OF CHROMOSOME9P21 AND Pl61NK4aSTATUS IN SPORADIC CUTANEOUS MELANoMk MioruTakata. Aki!dde Fuibnoto, Reiii Morita. Naohito Hat& Kazohiko TakehsrqLkpxbnent of Dermatology, Kanazawa University School of Medicine, Kanazawa, Japan. To examine the involvement of chromosome 9~21 loss and inactivation of a putative turnour suppressor gene pl6JNKk in tumorigenesis and metastatk progression of sporadic melanoma, we carried out PCR-based LOH analysis using 9 miaosateliite markers l!ml mapped to 9p21, direct sequencing and methyl&on-specific PCR (MSP) analyses of the pl6~~ gene, and bnmunohistochemistry for p16INK4a protein in 49 primary tomows and 13 corresponding metastascs. LQH of 9p21 was detected in 12 (24%) primary tomoors, all but one oossiblv. encomnassine oldmK4* locus. However. . I‘ direct sequen&g of exons 1 and 2 of the plmK& gene revealed only one heterozygous mutation at codon 81 (CCC->cTc; Pro->Leu) although two novel germlie polymorphisms were found. No methyl&ion of CpG islands of thepl@r%da gene was detected in 10 primary turnours showing 9p21 LOH where MSP wss successful. Patterns of 9p21 allelk loss were completely identical in 6 pairs of primary tunou and corresponding metastases. No additional pl6lNKh mutations were found in all 13 m&static tomours. whereas complete loss of p161NKhexpression was observed in 2 metastases. These results suggest that 1) mutation and tm”scriptional inactivation (due to aberrant methvlation of CoG islands) of the olmK+a eene are rare in turnours showing 9p21 l&s (thus th& may be &her t&our s”p&ssor genes in this region), 2) LQH of 9p21 is a” early event occwring before m&static clones diverge, and 3) loss of pl0NKh expression may be aswciated with metastaticprogression.
0291
0294
MITOCHONDRSALDNA DELEI’IONS ARE COMMON IN BPS+ AND SQUAMOUS CELL CARCINOMAS. w&i?, Depts. of Dennatol. and MoBSA The purpose of this shldy war to determine the frequency of mitcchondxia! chromosomal deletions in normal ski” and in squamous cell (SCC) and basal cell @CC) carcinomas. Mitocbondria repre.scntthe energy pow&owe of the cell but as a conseqwce produce a tremendous amount of reactive oxygen species (ROS). I” addition to the i”binskaUy produced ROS, kexatinccytesm”st deactivate ROS prodowl by ex”osure to “llrwiokt fIJV) radiatio”. Oxidative stress is induced when “moxid”“ts c&J the capacity of rhe a&oxidant “lecha”is”ls to detoxify Iban. Pote-“liallong term consequences of oxidative include cell damage, aping, cell death and ca”cer. Mitocho”drial DNA (mlDNA) is a 16.569 nuekotide double stranded circular g&c element located in !he cytoplasm. mtDNA is more sensitive !ha” nuclear DNA to the effects of W induced oxidative damage. Mutations produced in mtDNA are not readily repaired and as a result deletions of various “ocle&ie lengths 00~~. Downstream effects of the mtDNA deletions include inefficient oxidwive phosphorylation and incmawd prvduction of ROS. In this study we.evaluted 12 samples of normal ski” surroundi” skin from pwdominateiy older individuals as well as 18 BCCs and 15 SCCs for the presence of mtDNA deletions using long extension KR to amplitj the entire mitochondrial genome from harvested gewmic DNA Opposing primers were located in the cytochrome b gene. Reaction producu were ekctmpboresed on agarose gels and evaluated for deletions by size. The mitochondrisl origin of the FCR products WBS confimwd by southern bloaing. 66% of the normal skin samples displayed mtDNA deletions as did > 90% of the SCCs. Interestingly only 33% of the BCCs showed mrDNA deletions. We conclude that mtDNA deletions arc common in nommI skin in the region of ski” cancers as well as in the BCCs and SCCs. We hypothe.sizet!ut these deletions are induced in part by ulhwiolet radiation and play a mechanistic mk in skim canzcr by increasing oxidative strers.
ISOLATION. C!+ARACTER!ZATION. AND !ZXPRESSION OF MURINE MUCl8 cDNA GENE. H. Yaw. M.-W. H. Wu. M.D. Sarradet. J.C. Ansel. CA. Annstrone G.-J. Wu. Departments of hlkmb!!kgy 8 Immunology and Dermatology, Emory Universi!y School of Medicine, Atlanta, GA, USA. !I has been proposed that a human adhesion molecule MUCIE (huMUC18 or Mel-CAM) plays an important pathogenic role in metasiatic melanoma pmgresskn. Using a nude mouse model, huMUC18 was found to promote the metastatic adiviIy of human melanoma cells lines. However, this mode! is limited by the use of a xenographk system in an immune deficient animal. To establish a syngeneic, immunocompetent mouse mode! for studvka the mechanism of induction of metaslalk pipens!!y of murine melanoma tits by MUClfJ, we have used the methods of RT-PCR and RACEPCR lo amp!!! and isolate the mouse cDNA cormsoondino to huMUC18. We deteanlned the seauenee of the murine MUCIB (muMUC18jcDNA. and found that this gene contains 28 nucleotides of 5’4TR and 1950 nucleotides of sequence encoding a pmtein of 850 amino acids. The codino seouence of the muMUCltl has 75% nudeotide and 76% amino acid aentity to’huMUCl8. Northern bkl studies of four different murine melanoma cell lines demonstrated that higher levels of muMUCl6 are expressed in cell lines with the greatest metastatic adwity suggesting that muMUC18 may have a pathogenic role in the metastasis of murine melanoma cells. Thus, we have identified the mudne counterpart of huMUC18 which will allow us to experimenta!~ detenine the role of this molecule in the malastatic activity of malignant melanoma and could lead to new approaches for preventing pmgresskn of this neoplasm.
mediatedexpressionof hMSH-2end p53 in the SCC cell line SCL-l by meansof RT-PCR. Heterogeneous hMSH-2 immunoreachity was detected in most samples analyzed.
0289
0292
SPECIFIC DETECTION OF MELANOMA CELLS IN THE BLOOD BY ML4 RT-PCR I, w S ,
MI’IXKHONDRIALDNA DELETIONS ARE COMMON IN BASAL AND SQUAMOUS CELL CARCINOMAS. p w Depts. of Demmtol. and Molecular Medicine. Emory Univ., Atkmta GA, USA The purpose of thii study was to dotennine the frequency of mitochondrial chranawmal deletions in normal skin and in s(~“a”touscell (SC0 and basal cell !BCC) carcinomas. Mitocbontia represe”t the energy’powerho”se&fthe’ceI1but as a consequence produce a tremendous amount of reactive oxygen species (ROS). I” additkm to the i”“i”sically produced ROS, keratinocytes must deactivate ROS pmd”ced by expo.we to ulaaviokt (UV) radiation. Oxidative stress is induced when prooxidants exceed me capacity of me a”tioxida”t mecha”is”ls u) detoxify than. Pote”l!al long teml cansequenees of ox&rive siress include .&I damage, a&8. cell death and cancer. Mitochoodrial DNA (mrDNA) is a 16569 “uckotide double strmded circular genetic element located in the cytoplasm. mtDNA is more sensitive than nuclear DNA to the effects of UV induced oxidative damage. Mutations pmdwd in mtJJNA are not readily repaired and as a result deletions of various mxle&i& le”8th.soccw. Downstream effects of the mtDNA deletions include ineffiiient oxidative phosphorylation and inneased production of ROS. In this study we evaluated 12 samples of nomul skin surrounding skin canfrom predominately older individuals as well as 18 BCCs and 15 SCCs for the presence of mtDNA deletions using long extension PCR to mnplii the entire mitochondrial genome from harvested genomic DNA opposing primers were located in the.cytochrome b gene. Reaction products were electmpboresed on agamse gels and evaluated for deletions by size. The mitcchondria? ori@” of the K!R pmducu wa( confirmed by southern blotting. 66% of the nommi skm samples displayed mfDNA deletions as did > 90% of the SCCs. Interestingly only 33% of the BCCs showed mtDNA deletions. We conclude that mtDNA deletions a comma” in normal ski” in the region of skin cancers as well as in the BCCs and SC& We hypot!xsiLe that these deletion are induced in part by “lwviolet radiation and play a mechanistic role in skin callfer by increasing axidative stress.
0290
0293
EXPRESSION AND REGULATION OF THE h4!SMATCH-REPAIR-ENZYME HMSH2 IN HUMAN SKIN TUMORS. 1. Reichrath’. K. Rsss’. P. Go&vein’. C. Welte?. M. S&f&, K.D. Zanr’ and W. Tileen’. Departments of ‘Dermatology and ‘Human Genetics, “niverwty of the Saarland, Homburg, Germany. Mutations in the coding regions of methyl-directed nusmatch-repair genes, particularly LMSH-2, are involved in microsatellite instability and therefore seem to play a crucial role in hereditary nonpolyposis co!orecta! cancer. As we could previously demonstrate a phynmlogical connection between the W-inducible tumor-suppressor ~53 and LMSH-2 by exploring a site with homology to the ~53 consensus sequence in the promotor @on of hh&H-2, we now studied thekxpresslon and regulation &hMSH-2 in human skin tumors. We analyzed immunoh~sttochemically the expression of hMSH-2, ~53 and the proliferation marker Ki-67 on paraffin sections of normal human skin end different epithclial skin rumours [malignant melanoma, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), Bowen’s disease and actinic kemtoses]. Furthermorewe investigated the UVB-
Expression of hMSH-2 was upregulated in all malignant skin tomours, most markedly tn BCCs and Bowen’s disease, as compared to normal human skin. HMSH-2 up-ngulat~on was mostly pronouncedin turnoursrevealing strong p53 immunoreactivity Expression analysis by means of RT-PCR m UVB-treated SCL-I cells revealed an induction of p53 mRNA and subsequent up-regulation of hMSH-2 mRNA in vitro. Our findings indzate that LMSH-2 is strongly expressed in malignant skin tumors, most markedly in those which reveal strong pS3 immunoreacfivity. The functional expression-analysis pomts to the fact that LMSH-2 is directly mvolved in ~53 mediated DNA-repair following UVBdamage.
LOSS OF HETEROZYGOSITY OF CHROMOSOME9P21 AND Pl61NK4aSTATUS IN SPORADIC CUTANEOUS MELANoMk MioruTakata. Aki!dde Fuibnoto, Reiii Morita. Naohito Hat& Kazohiko TakehsrqLkpxbnent of Dermatology, Kanazawa University School of Medicine, Kanazawa, Japan. To examine the involvement of chromosome 9~21 loss and inactivation of a putative turnour suppressor gene pl6JNKk in tumorigenesis and metastatk progression of sporadic melanoma, we carried out PCR-based LOH analysis using 9 miaosateliite markers l!ml mapped to 9p21, direct sequencing and methyl&on-specific PCR (MSP) analyses of the pl6~~ gene, and bnmunohistochemistry for p16INK4a protein in 49 primary tomows and 13 corresponding metastascs. LQH of 9p21 was detected in 12 (24%) primary tomoors, all but one oossiblv. encomnassine oldmK4* locus. However. . I‘ direct sequen&g of exons 1 and 2 of the plmK& gene revealed only one heterozygous mutation at codon 81 (CCC->cTc; Pro->Leu) although two novel germlie polymorphisms were found. No methyl&ion of CpG islands of thepl@r%da gene was detected in 10 primary turnours showing 9p21 LOH where MSP wss successful. Patterns of 9p21 allelk loss were completely identical in 6 pairs of primary tunou and corresponding metastases. No additional pl6lNKh mutations were found in all 13 m&static tomours. whereas complete loss of p161NKhexpression was observed in 2 metastases. These results suggest that 1) mutation and tm”scriptional inactivation (due to aberrant methvlation of CoG islands) of the olmK+a eene are rare in turnours showing 9p21 l&s (thus th& may be &her t&our s”p&ssor genes in this region), 2) LQH of 9p21 is a” early event occwring before m&static clones diverge, and 3) loss of pl0NKh expression may be aswciated with metastaticprogression.
0291
0294
MITOCHONDRSALDNA DELEI’IONS ARE COMMON IN BPS+ AND SQUAMOUS CELL CARCINOMAS. w&i?, Depts. of Dennatol. and MoBSA The purpose of this shldy war to determine the frequency of mitcchondxia! chromosomal deletions in normal ski” and in squamous cell (SCC) and basal cell @CC) carcinomas. Mitocbondria repre.scntthe energy pow&owe of the cell but as a conseqwce produce a tremendous amount of reactive oxygen species (ROS). I” addition to the i”binskaUy produced ROS, kexatinccytesm”st deactivate ROS prodowl by ex”osure to “llrwiokt fIJV) radiatio”. Oxidative stress is induced when “moxid”“ts c&J the capacity of rhe a&oxidant “lecha”is”ls to detoxify Iban. Pote-“liallong term consequences of oxidative include cell damage, aping, cell death and ca”cer. Mitocho”drial DNA (mlDNA) is a 16.569 nuekotide double stranded circular g&c element located in !he cytoplasm. mtDNA is more sensitive !ha” nuclear DNA to the effects of W induced oxidative damage. Mutations produced in mtDNA are not readily repaired and as a result deletions of various “ocle&ie lengths 00~~. Downstream effects of the mtDNA deletions include inefficient oxidwive phosphorylation and incmawd prvduction of ROS. In this study we.evaluted 12 samples of normal ski” surroundi” skin from pwdominateiy older individuals as well as 18 BCCs and 15 SCCs for the presence of mtDNA deletions using long extension KR to amplitj the entire mitochondrial genome from harvested gewmic DNA Opposing primers were located in the cytochrome b gene. Reaction producu were ekctmpboresed on agarose gels and evaluated for deletions by size. The mitochondrisl origin of the FCR products WBS confimwd by southern bloaing. 66% of the normal skin samples displayed mtDNA deletions as did > 90% of the SCCs. Interestingly only 33% of the BCCs showed mrDNA deletions. We conclude that mtDNA deletions arc common in nommI skin in the region of ski” cancers as well as in the BCCs and SCCs. We hypothe.sizet!ut these deletions are induced in part by ulhwiolet radiation and play a mechanistic mk in skim canzcr by increasing oxidative strers.
ISOLATION. C!+ARACTER!ZATION. AND !ZXPRESSION OF MURINE MUCl8 cDNA GENE. H. Yaw. M.-W. H. Wu. M.D. Sarradet. J.C. Ansel. CA. Annstrone G.-J. Wu. Departments of hlkmb!!kgy 8 Immunology and Dermatology, Emory Universi!y School of Medicine, Atlanta, GA, USA. !I has been proposed that a human adhesion molecule MUCIE (huMUC18 or Mel-CAM) plays an important pathogenic role in metasiatic melanoma pmgresskn. Using a nude mouse model, huMUC18 was found to promote the metastatic adiviIy of human melanoma cells lines. However, this mode! is limited by the use of a xenographk system in an immune deficient animal. To establish a syngeneic, immunocompetent mouse mode! for studvka the mechanism of induction of metaslalk pipens!!y of murine melanoma tits by MUClfJ, we have used the methods of RT-PCR and RACEPCR lo amp!!! and isolate the mouse cDNA cormsoondino to huMUC18. We deteanlned the seauenee of the murine MUCIB (muMUC18jcDNA. and found that this gene contains 28 nucleotides of 5’4TR and 1950 nucleotides of sequence encoding a pmtein of 850 amino acids. The codino seouence of the muMUCltl has 75% nudeotide and 76% amino acid aentity to’huMUCl8. Northern bkl studies of four different murine melanoma cell lines demonstrated that higher levels of muMUCl6 are expressed in cell lines with the greatest metastatic adwity suggesting that muMUC18 may have a pathogenic role in the metastasis of murine melanoma cells. Thus, we have identified the mudne counterpart of huMUC18 which will allow us to experimenta!~ detenine the role of this molecule in the malastatic activity of malignant melanoma and could lead to new approaches for preventing pmgresskn of this neoplasm.
mediatedexpressionof hMSH-2end p53 in the SCC cell line SCL-l by meansof RT-PCR. Heterogeneous hMSH-2 immunoreachity was detected in most samples analyzed.