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BLC-2 antisense oligonucleotides inhibit merkel cell carcinoma growth in SCID mice
s135
ESDR I JSID I SID Abstracts
0808
0805 RT-PCR
FOR
TYROSfNASE-mRNA
EVALUATION STRATEGY MARKERS
lh’ 123 MELANOMA
AND
POSITfVE CORRELATION
CELLS WITH
IN
PERJPHERAL KNOWN
BLOOD
PROGNOSTIC
PATIENTS
RTPCR for the detection of tyrosinasemRNAqositwecells in penpherslbloodof melsooms oatimts as a oosstblemarkerofhematoneoousdissemmationbaa demonstratedvarwta detectton presentstudy examined the &sitivny and reproducibthty of the techmoue t&g a
rates The
protocol of multiple PCR reactions to determine ctrculatiog melenccytic cells For each of the 123 melaooina patients utcludedto tbts study,4 nestedPCRs wre perfomtedfrom 2 blcod spwitnensrequirmgboth PCP.sfrom at least one bloodsampleto be positive for coosidetinga pattentas posttive.Thus, a definitiveresult was obtainedto 98%of the cases,whereasonly 1.6% lackedconcluswefind&s. Thus, we founda correlaticmbetweeotyrosioasedetectioorate sod cluucal stage Ctrculatin8tyrosmase-mRNApositive cells ~ieredetectedin 13%of pabentswith primaq tumor. 17%with regionalski&mph-node metastasisand 44% wtb distant tuetastasa Positiwtyalso correlatedwttb knowntnelaoomaprogressionmarkerssuch as gender,tuotor tickness and histolc&al type Positweresultswereobtamedmorefrequently(a) to atenthan m women,(b) in patieotswiththick primarymelaoomas(> 4 mm 38%)than to thosewiththinner tuotors(1.1-4mm: 22%, < 1m 5%). sod(c) m pattents wah ooo-classrtiable (38%).ocdular (34%)sod occultpnmary melsoomas(30%)ccmparedto those wtb acmlentiginous(17%), superficialspreading(9%)or lentigomalignamelanoma(0%) Thesefindingssuggestthat detectionof tyrosiosse-mpNA-posttlve cells in penpberalbloodis not an adequafemarkerfor &ntifyvlg melaoomapatieotswtb dntant metastasis RT-PCRposibvity in early melanoma stages, however, as correspondrug to other prognostic paraoxters, may ituhcete increased risk for the developmeot of hematogeoous metastasis and may be of value as a progression marker
BCLJ ANTISENSE OLIGONUCLEOTIDES INBIBIT MERKEL CELL CARCINOMA GROWTH IN SCID MICE H. Schlaabauer-Wadl’z, I. Malls, S. Waltering4, H.-G. Eichler”, K. Wolf?, H. Pehatnberaer’, 8. lansen’~’Dept. of DennatologyiDiv. of General Dermatology’, Dept. of Clinical Phartnacologyr, Vienna, Austria. Dept. of Dermatology, University Hospital Eppendorf. Hamburr, Germany. Dem. of Dertnatolow’. Mannheim. Germrmv Merkel cell carcinoma is a rare, highly aggressiv~n&endocrihk skin &nor with the potential to metastasize. In contrast lo fetal end especially adult Merkel cells, Merkel cell carcinoma expresses high levels of bcl-2. Capable of blocking programmed cell death, beI-2 has been shown to play an important role in cell turnover and also in chemoresistance in certain systems. High bcl-2 expression may be of importance in the biological and clinical behavior ofMerke1 cell carcinoma. In a newly established SCID mouse xenotransplantation model for human Merkel cell carcinoma we investigated the influence of bcl-2 antisenae oligonucleotides on tumor growth compared to control oligonucleotides or Cisplatin. Only bcl-2 antisense treatment resulted in a dramatic reduction of tumor growth or even complete remission whereas control oliganucleotides or Cisplatin had no significant an&tnor effect compared to saline treated controls. Tlx contributions of seouence soecific antisense . . effects as well as possible sequence dependent and sequence independent non-antisense interactions that may contribute to this dramatic reduction in tumor growth are currently under investigation. In any case, treatment of human Merkel cell carcinoma with antisense bcl-2 oligonucleotides seems to be a therapeutic concept worth pursuing.
0806
0809
REGULATION OF EPIDEPMAL DfFFERENTIATION COMPLEX GET EXPP.ESSfON BY AGENTS THAT ALTER CHROMATTN S’fW.JCT”RF If F&r ax, XP zhne fkpsmnents of Dertastofoay and tftadiation Oncology (Cancer Biology), Uwersuy of Michigan,Ann Arbor, MI TIE ep*ermd differe”oatmn cmn*lex (EDC), loca,ed 0” chromaaomal band lq21. CO”SiS,Sof at least 25 genes that undergo coordinate regulation during keminccyte d~fferenhatwn. including memberr Of the SIW an*small protine nch rearon (SPRR, gene clusters. The reason (If any) We have mvesugsted the hypothesis for the chutered orgsoizsuan of these genes Is u*own. that a general opening of chromsttn structure might occur durmg kerstisocyte drffereabstion. resulting in a generai up-regulatron of EDC genes. Ssoodq cultures of norrust human keratinwyter @H(K) were grown in mcditied MCDBl53 and H&!&f cells were grown in DM!Zvf,lOB FCS. Cells were treatedfor “srious times wrul two agents ksawn 10 promote ‘“open”chromatin smzcture: 5-wxcytidine (5AC. I-1W pM), which mhibiu CpC reethykrse and sodium butyrste @&It. t-10 r&f). sduch inhibits histone deacetytsse. Using Northern blatting, we measured the expression ofCaN (SILL, AIOOAZ). s mcmberofthc St00 gene cluster, SPRRl sod 2. members of the SPRR gene cluster,anduwolucrln(WV).which is Both agents structuralty related to the SPRR genes. 36B4 served s.s a loading conlrol markedly increasederpression of SPRRl/Z (detected using a mixedprobe)andw rn NHK.
POSITIVE REGULATlON OF PlS (INK4B). A TUMOR SUPPRESSOR GENE. lN MELANOMACELL LlNES BY TETP.ACYCilNE. T.R. Pacheco.S. Na, D.A. N,,. W. Weston and LH. Maxwell. Departmentof Dermatoloev, Umversih, of Colorado Health SciencesCenter, Denver, Colorado80262,USA. Identification of frequent deletions in melanoma kindreds and cell lures of the cyclin dependentkioase inhibitors,pl5 and ~16, locatedon Cbr 9~21 have stimulatedinterest in the relative function of these homologous potential tumor suppressors. Two tetracycline regulatory systems (TET-OFFsod TET-ON)(Gossen M. Science 268:1766 (1995)) have be&r used to modulate expression of transfe&d genes. Though the TET-ON system is technicallydifficult, our laboratw utilbed it becauseof its inherentadvsnteues. In order to examine ~15 function in melanomacells, we trsnsfectedthe ceil lines, WM1617 sod A375, with the TET-ONtqulatory system sod pEBO-To-~15,an sutonomousplasmid enabling TET controlledexpressionof human~15 cDNA. Westernanalysis showedthat the parental melaooma cell lines retain Rb expression without expressing pl5 or ~16. These treosfectedcells abundantlyexpressedpl5 following doxycyclineinduction (I p&n) with
w,ti the gnstest responsesobserwdat 1 PM 5AC and1 mM NC,. In msr*edcoma%
barely detectable basal expression in doxycycline’s absence. lmmunostsioing identified cytoplssmic pl5 expression on so individual cell basis and showed enhanced expression efter addition of doxycycline. Flow cytometric analysis of cell cycle parameters showed GO-GI phase accumulation when pl5 expression is induced by doxycycline. Forty-eight hours after
expressionof CsNt9 wss markedly reducedby both agents. Basal levels of both SPRR ind tNV inneared ss cells low-calcium NHK became more connuent, and after shifting NHK subconfluent NHK to high-calcium DMEM/ 1046FCS. At cotiuenee, low-calcium bccamcrefractory to NaB, but not to SAC. HaCaT cells exprssed much tower bass, levels of dt three usescopts. and were markedly less responsive to 5AC and NaB. Our nsuhs are not consistent with a simple global activauon of the EDC in response to agents whtch “open” chromstin suuctwe. They do. however, indirectly suppan B role for histone scetylaoon in the contml of FDC genes dunng KC drfferentiatfon.
To our knowledge,this is the first use of the PET-ONsystem lo melanomacell lines. This systemwill facilitatestudies of the role of pl5 on anchorage-independent growth, induction of apoptosis, and expression of othercell cycleregulatoryproteinsin melanomacell lines.
0807
0810
Identification of cyclosporin A-responsive genes in human keratinocyes by mRNA differential display M. 0~0. I. Suzuki. T. Soma. T. Takahaahi, T. Hibino and Y. Nakavama Shiaeido Research Center, Yokohama, Japan. In order to obtain a molecular Insight of the hypertrichosis caused by cyclosporin A @A), we investigated the effect of CysA using organ and cell culture systems. Lower concentration of CysA (0.1, 1.0 @ml) accerelated the elongation of hair shaft and hair follicle significantly. But higher concentration (10 @ml) suppressed the elongation accompanying the morphological disorganization. RT-PCR showed at least three types of cyclophilins[CysA binding protein (Cyp)]were expressed in human keratinocytes (KC), fibroblasts, outer root sheath cells (OR’S) and dermal papilla cells (DP), suggesting possible involvment of tbese cells in hair growth. Interestingly, expression of CypD was detected only in KC, whereas, Cyp40 expression is restricted to ORS. In the next step, we tried to isolate the CysA responsive gene8 in human KC and DP using differential mRNA display. Among the candidates obtained, we confirmed that expression of RTP, whose function ia unknown, and follistatin an inhibitor of activin, was affected only in KC by CyaA. R’IP expression was induced in KC after 24 hr of CysA treatment, while the expression of follistatin was suppressed in the presence of high dose of CysA (10 &nl). At this concentration, the growth of KC was remarkably suppressed. The expression of follistatin seemed to correlate to proliferation of KC, since it became no longer detected in KC after they reached the confluent condition. In situ analysis showed that follistatin was expressed in both epithelial and mesencymal cell in hair follicle, suggesting that CysA plays a role by regulating the activin pathway.
TRANSCRIPTION FACTOR UBBPS IN RHINO MOUSE EPI~E,RMIS IS UPREGULATED BY FTINOL BUT NOT RBTINOIC ACID. E&bard A. Romero. &lvador Gona&ea. and EdwardV. Mavtin lWelhnan Laboratories of Photomedicine, Department of Dermatology, “Departmentof Medicine (Endocrinology), Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Transcription factors of the CCAATcnhancer binding protein family (UBBPs), identified as important regulators of differentiation-specific genes in liver and adipose tissues, have recently been found in the skin. The most abundant of these factors, UBBPS, can transactivate the keratin 10 (KlO) gene in vitro, but its role in regulating epidermal differentiation remains unproven. Here, we used topical retinoids to modii the balance between epidennal proliferation and dtffetentiation. m order to investigate the effects upon expression of the C/EBPs in the epidermis of the rhino mouse. Mice were randomly divided into five different groups. The following agents were applied to the back daily for five days per week over two weeks, ethanoVpropyleneglycol as vehicle, all-tram retinoic acid (RA. 0.025,O.Ml25,O.CGU25%), andretinol (ROL, 0.025%). Levels of three C/BPBs (C/BBPa, C&BPS, and CHOP) and of other markers involved in epidennal cell kinetics (KlO, involucrin, and PCNA) were analyzed by in situ immunoqtochemisny from paraftin~mbedded skm sections. As expected, ROL and RA showed an epidennal hyperplastic response; however, only ROL showed increased expression of KlO and lnvolucrln. A highly significant upregulation of UBBPS, evidenced by strong nuclear expression witbin the basal and suprabasel layers, was induced by ROL but not by RA. increased levels of CHOP, both nuclear and cytoplasmic, were found with ROL and 0.025%&4. No changes in C/BBPa levels were observed in any of the treated groups. In summary, the demonstration of a strong nuclear induction of UBBpP by ROL, preferentially over RA, suggests an imponarn physiologic role for retinol in the regulation of genes involved in eptdermal growth and differentiatton in viva.
doxycycline induction, 67% of the transfected cells accumulated opposed to 40% of the trsnsfected control cells.
in the GO-GI
phase as
ESDR I JSID I SID Abstracts
0808
0805 RT-PCR
FOR
TYROSfNASE-mRNA
EVALUATION STRATEGY MARKERS
lh’ 123 MELANOMA
AND
POSITfVE CORRELATION
CELLS WITH
IN
PERJPHERAL KNOWN
BLOOD
PROGNOSTIC
PATIENTS
RTPCR for the detection of tyrosinasemRNAqositwecells in penpherslbloodof melsooms oatimts as a oosstblemarkerofhematoneoousdissemmationbaa demonstratedvarwta detectton presentstudy examined the &sitivny and reproducibthty of the techmoue t&g a
rates The
protocol of multiple PCR reactions to determine ctrculatiog melenccytic cells For each of the 123 melaooina patients utcludedto tbts study,4 nestedPCRs wre perfomtedfrom 2 blcod spwitnensrequirmgboth PCP.sfrom at least one bloodsampleto be positive for coosidetinga pattentas posttive.Thus, a definitiveresult was obtainedto 98%of the cases,whereasonly 1.6% lackedconcluswefind&s. Thus, we founda correlaticmbetweeotyrosioasedetectioorate sod cluucal stage Ctrculatin8tyrosmase-mRNApositive cells ~ieredetectedin 13%of pabentswith primaq tumor. 17%with regionalski&mph-node metastasisand 44% wtb distant tuetastasa Positiwtyalso correlatedwttb knowntnelaoomaprogressionmarkerssuch as gender,tuotor tickness and histolc&al type Positweresultswereobtamedmorefrequently(a) to atenthan m women,(b) in patieotswiththick primarymelaoomas(> 4 mm 38%)than to thosewiththinner tuotors(1.1-4mm: 22%, < 1m 5%). sod(c) m pattents wah ooo-classrtiable (38%).ocdular (34%)sod occultpnmary melsoomas(30%)ccmparedto those wtb acmlentiginous(17%), superficialspreading(9%)or lentigomalignamelanoma(0%) Thesefindingssuggestthat detectionof tyrosiosse-mpNA-posttlve cells in penpberalbloodis not an adequafemarkerfor &ntifyvlg melaoomapatieotswtb dntant metastasis RT-PCRposibvity in early melanoma stages, however, as correspondrug to other prognostic paraoxters, may ituhcete increased risk for the developmeot of hematogeoous metastasis and may be of value as a progression marker
BCLJ ANTISENSE OLIGONUCLEOTIDES INBIBIT MERKEL CELL CARCINOMA GROWTH IN SCID MICE H. Schlaabauer-Wadl’z, I. Malls, S. Waltering4, H.-G. Eichler”, K. Wolf?, H. Pehatnberaer’, 8. lansen’~’Dept. of DennatologyiDiv. of General Dermatology’, Dept. of Clinical Phartnacologyr, Vienna, Austria. Dept. of Dermatology, University Hospital Eppendorf. Hamburr, Germany. Dem. of Dertnatolow’. Mannheim. Germrmv Merkel cell carcinoma is a rare, highly aggressiv~n&endocrihk skin &nor with the potential to metastasize. In contrast lo fetal end especially adult Merkel cells, Merkel cell carcinoma expresses high levels of bcl-2. Capable of blocking programmed cell death, beI-2 has been shown to play an important role in cell turnover and also in chemoresistance in certain systems. High bcl-2 expression may be of importance in the biological and clinical behavior ofMerke1 cell carcinoma. In a newly established SCID mouse xenotransplantation model for human Merkel cell carcinoma we investigated the influence of bcl-2 antisenae oligonucleotides on tumor growth compared to control oligonucleotides or Cisplatin. Only bcl-2 antisense treatment resulted in a dramatic reduction of tumor growth or even complete remission whereas control oliganucleotides or Cisplatin had no significant an&tnor effect compared to saline treated controls. Tlx contributions of seouence soecific antisense . . effects as well as possible sequence dependent and sequence independent non-antisense interactions that may contribute to this dramatic reduction in tumor growth are currently under investigation. In any case, treatment of human Merkel cell carcinoma with antisense bcl-2 oligonucleotides seems to be a therapeutic concept worth pursuing.
0806
0809
REGULATION OF EPIDEPMAL DfFFERENTIATION COMPLEX GET EXPP.ESSfON BY AGENTS THAT ALTER CHROMATTN S’fW.JCT”RF If F&r ax, XP zhne fkpsmnents of Dertastofoay and tftadiation Oncology (Cancer Biology), Uwersuy of Michigan,Ann Arbor, MI TIE ep*ermd differe”oatmn cmn*lex (EDC), loca,ed 0” chromaaomal band lq21. CO”SiS,Sof at least 25 genes that undergo coordinate regulation during keminccyte d~fferenhatwn. including memberr Of the SIW an*small protine nch rearon (SPRR, gene clusters. The reason (If any) We have mvesugsted the hypothesis for the chutered orgsoizsuan of these genes Is u*own. that a general opening of chromsttn structure might occur durmg kerstisocyte drffereabstion. resulting in a generai up-regulatron of EDC genes. Ssoodq cultures of norrust human keratinwyter @H(K) were grown in mcditied MCDBl53 and H&!&f cells were grown in DM!Zvf,lOB FCS. Cells were treatedfor “srious times wrul two agents ksawn 10 promote ‘“open”chromatin smzcture: 5-wxcytidine (5AC. I-1W pM), which mhibiu CpC reethykrse and sodium butyrste @&It. t-10 r&f). sduch inhibits histone deacetytsse. Using Northern blatting, we measured the expression ofCaN (SILL, AIOOAZ). s mcmberofthc St00 gene cluster, SPRRl sod 2. members of the SPRR gene cluster,anduwolucrln(WV).which is Both agents structuralty related to the SPRR genes. 36B4 served s.s a loading conlrol markedly increasederpression of SPRRl/Z (detected using a mixedprobe)andw rn NHK.
POSITIVE REGULATlON OF PlS (INK4B). A TUMOR SUPPRESSOR GENE. lN MELANOMACELL LlNES BY TETP.ACYCilNE. T.R. Pacheco.S. Na, D.A. N,,. W. Weston and LH. Maxwell. Departmentof Dermatoloev, Umversih, of Colorado Health SciencesCenter, Denver, Colorado80262,USA. Identification of frequent deletions in melanoma kindreds and cell lures of the cyclin dependentkioase inhibitors,pl5 and ~16, locatedon Cbr 9~21 have stimulatedinterest in the relative function of these homologous potential tumor suppressors. Two tetracycline regulatory systems (TET-OFFsod TET-ON)(Gossen M. Science 268:1766 (1995)) have be&r used to modulate expression of transfe&d genes. Though the TET-ON system is technicallydifficult, our laboratw utilbed it becauseof its inherentadvsnteues. In order to examine ~15 function in melanomacells, we trsnsfectedthe ceil lines, WM1617 sod A375, with the TET-ONtqulatory system sod pEBO-To-~15,an sutonomousplasmid enabling TET controlledexpressionof human~15 cDNA. Westernanalysis showedthat the parental melaooma cell lines retain Rb expression without expressing pl5 or ~16. These treosfectedcells abundantlyexpressedpl5 following doxycyclineinduction (I p&n) with
w,ti the gnstest responsesobserwdat 1 PM 5AC and1 mM NC,. In msr*edcoma%
barely detectable basal expression in doxycycline’s absence. lmmunostsioing identified cytoplssmic pl5 expression on so individual cell basis and showed enhanced expression efter addition of doxycycline. Flow cytometric analysis of cell cycle parameters showed GO-GI phase accumulation when pl5 expression is induced by doxycycline. Forty-eight hours after
expressionof CsNt9 wss markedly reducedby both agents. Basal levels of both SPRR ind tNV inneared ss cells low-calcium NHK became more connuent, and after shifting NHK subconfluent NHK to high-calcium DMEM/ 1046FCS. At cotiuenee, low-calcium bccamcrefractory to NaB, but not to SAC. HaCaT cells exprssed much tower bass, levels of dt three usescopts. and were markedly less responsive to 5AC and NaB. Our nsuhs are not consistent with a simple global activauon of the EDC in response to agents whtch “open” chromstin suuctwe. They do. however, indirectly suppan B role for histone scetylaoon in the contml of FDC genes dunng KC drfferentiatfon.
To our knowledge,this is the first use of the PET-ONsystem lo melanomacell lines. This systemwill facilitatestudies of the role of pl5 on anchorage-independent growth, induction of apoptosis, and expression of othercell cycleregulatoryproteinsin melanomacell lines.
0807
0810
Identification of cyclosporin A-responsive genes in human keratinocyes by mRNA differential display M. 0~0. I. Suzuki. T. Soma. T. Takahaahi, T. Hibino and Y. Nakavama Shiaeido Research Center, Yokohama, Japan. In order to obtain a molecular Insight of the hypertrichosis caused by cyclosporin A @A), we investigated the effect of CysA using organ and cell culture systems. Lower concentration of CysA (0.1, 1.0 @ml) accerelated the elongation of hair shaft and hair follicle significantly. But higher concentration (10 @ml) suppressed the elongation accompanying the morphological disorganization. RT-PCR showed at least three types of cyclophilins[CysA binding protein (Cyp)]were expressed in human keratinocytes (KC), fibroblasts, outer root sheath cells (OR’S) and dermal papilla cells (DP), suggesting possible involvment of tbese cells in hair growth. Interestingly, expression of CypD was detected only in KC, whereas, Cyp40 expression is restricted to ORS. In the next step, we tried to isolate the CysA responsive gene8 in human KC and DP using differential mRNA display. Among the candidates obtained, we confirmed that expression of RTP, whose function ia unknown, and follistatin an inhibitor of activin, was affected only in KC by CyaA. R’IP expression was induced in KC after 24 hr of CysA treatment, while the expression of follistatin was suppressed in the presence of high dose of CysA (10 &nl). At this concentration, the growth of KC was remarkably suppressed. The expression of follistatin seemed to correlate to proliferation of KC, since it became no longer detected in KC after they reached the confluent condition. In situ analysis showed that follistatin was expressed in both epithelial and mesencymal cell in hair follicle, suggesting that CysA plays a role by regulating the activin pathway.
TRANSCRIPTION FACTOR UBBPS IN RHINO MOUSE EPI~E,RMIS IS UPREGULATED BY FTINOL BUT NOT RBTINOIC ACID. E&bard A. Romero. &lvador Gona&ea. and EdwardV. Mavtin lWelhnan Laboratories of Photomedicine, Department of Dermatology, “Departmentof Medicine (Endocrinology), Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Transcription factors of the CCAATcnhancer binding protein family (UBBPs), identified as important regulators of differentiation-specific genes in liver and adipose tissues, have recently been found in the skin. The most abundant of these factors, UBBPS, can transactivate the keratin 10 (KlO) gene in vitro, but its role in regulating epidermal differentiation remains unproven. Here, we used topical retinoids to modii the balance between epidennal proliferation and dtffetentiation. m order to investigate the effects upon expression of the C/EBPs in the epidermis of the rhino mouse. Mice were randomly divided into five different groups. The following agents were applied to the back daily for five days per week over two weeks, ethanoVpropyleneglycol as vehicle, all-tram retinoic acid (RA. 0.025,O.Ml25,O.CGU25%), andretinol (ROL, 0.025%). Levels of three C/BPBs (C/BBPa, C&BPS, and CHOP) and of other markers involved in epidennal cell kinetics (KlO, involucrin, and PCNA) were analyzed by in situ immunoqtochemisny from paraftin~mbedded skm sections. As expected, ROL and RA showed an epidennal hyperplastic response; however, only ROL showed increased expression of KlO and lnvolucrln. A highly significant upregulation of UBBPS, evidenced by strong nuclear expression witbin the basal and suprabasel layers, was induced by ROL but not by RA. increased levels of CHOP, both nuclear and cytoplasmic, were found with ROL and 0.025%&4. No changes in C/BBPa levels were observed in any of the treated groups. In summary, the demonstration of a strong nuclear induction of UBBpP by ROL, preferentially over RA, suggests an imponarn physiologic role for retinol in the regulation of genes involved in eptdermal growth and differentiatton in viva.
doxycycline induction, 67% of the transfected cells accumulated opposed to 40% of the trsnsfected control cells.
in the GO-GI
phase as