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MTS2, and p18 genes in primary and metastatic lung cancer
Abstracts/Lung
Cancer 13 (1995) 81-104
Ca” rbd not a5ect the action potential production, whereas 5 iM TTX or subslttutton of Na* with cholineabolished rt. Action pMentiaIselicited rn the Ca”-free condition were reversibly blocked by 4 mM M&I, due to the Mn”-induced inhibition of voltage-dependent sodium currents (I(Na)) Therefore, Na’ channels, not Ca’* channels, underlie the excitability of SCLC cells. Whole-cell @a) was maximal with stepdepolarizing stimulations to 0 mV, and reversed at +45 2 mV, tn accord with the p=dicted Nemst ectuilibrium pofential for a Na+-selective channel. i(Na) evoked by dep&izing test potentials (-60 to +40 mV) exhibited a transient time course and activation/ mactivation kinetics typical of neuronal excitable membranes; the plot oftbe Hodgkin-Huxley parameters. m() and ho. also revealed biophysical similarity between SCLC and neumnal Na’ channels The single channel current amplitude, as nuasured with the inside-out patch co&guration. was I .O pi at -20 mV with a slope conductance of 12. I pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (L.ES), whtch are known to inhibit I(Ca) and I(Na) in bovine adrenal chmmaliin cells. also si8niticantly inhibited IlNa) in SCLC cells. These results indicate that (i)action po(endals in human SCLC cells nsult from the regenerative increase in voltage-gated Na* channel conductance: (ii) timdamental characteristics of SCLC Na’ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na’ channels are an additional target of the pathological IgG present in the patients’ sera
the armsense-treated cells compared tith control-treated cells in NCIH209. Togctkr with unique charaaeristia of the Llnyc gene. including: (a) a frequently amplified and overexpressed state in SCLC; and (b) very rcstictcd and low-level expression in human adult tissues. the present data indicate that L-myc is a good candidate for the target gene for antisenw DNA therapy based on molecular biological diagnosis in SCLC.
Mutations in the pl6(lNKJ)/MTSl/CDKN2, plS(INKdB)/ MTSZ, and pig genes in primary and metartatic lung cancer Okamoto A, Hussain SP, Hagiwara K, Spillare EA. Ruin MR. 1448-51. We exammed Ihe genomic status ofcychn&pmdent kinase-4 and -6 mlub~tors, pl6(INK4). pl5(INK4B). and ~18, I” 40 primary lung cancers and 31 metastatic lung cancers Alterations of the p16(INK4) gene were detected in 6 (2 wetirons and 4 homozygous deletions) of 22 metastat~ non-small cell lung cancers (NSCLCs: 27%). but none weredetected in 25 pnmay NSCLCs. 15 primarysmalloell lungcancers (SCLCs). or 9 metastatrc SCLCs. mdrcatin8 that mutation tn Ihe pl6(lNK4) gene IS a late event in NSCLC c&nogenesis Although three mtragenic mutationsofthc pIS(INK4B) gene were dctcctcd fin 25 prrmary NSCLCs (12%) and f,\e homozygous deletions of Ihe olS(INK4Bl eerie wcrc detected tn 22 NSCLCs 123%L no eenetx alteratiowofthepl5(INK4B) genewere foundin primary and metastauc SCLCs. The pl8 gene was wdd type tn these 7 I lung cancers. except I metastatic NSCLC whrch showed loss of hetcrwygowy We also esamincdaltcrat1onsofthcrthrcegennandespress~onofpl6(INK4) rn 21 human lung cancer cell hncs. Alteratmns of the plG(INK4) and plS(lNK4B) genes wcredetected I” 7l%ofthe NSCLC cell hnes (n = 14) and 50% of the NSCLC cell hncs (n = l4), rcspect~\cl>. but there were mane ,n the 7 SCLC cell hnes studwd No plR mutauons were dctcctedmthcsc21 cell hnes Thescraultr~nd~catethatbMhpl6lINK4~ andplS(INKJB)gene mutat,onsareasroc~rtcd~rth tumorprogrcsrmn ofa subset ofNSCLC. but not ofSCLC. and tbat plS(INK4B) mutatwns might also be an early event m the molecular pathogcncsrs of a subset or NSCLC .
Mechmims of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplican Eiidems EWHM. De Haa M Ccco-Martin JM. Dttenheim CPE. Zaman &I. DawerseHG et al. &ision Uo/ecu/ar Biology Netherlands Cancer Insdtofe, Plesmanlaan 121, 1066 CYAmstenlam Int I Cancer l995;60:676-84. Some mtdtidrugresistant cell lines over-express Ihe gene encoding the multidrug-resistann-associaled protein f.MRP). In all all lines reported thus far, over-expression is assccialed with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human Iuwcanccr all lines that myer a range ofdntrrcsistance lewls, and we&e attalyzed the MRP ampli&. In th;SW-l573derived. waklv resistant cell line 30.3M. h4RP mRNA IS elevated 3fold in & &s&x of gene amplification. Run-on analysis shows that the increased MRP gene expression in this cell line is due lo transcriptional activation. In the highly resistant GLC4/ADR and CO&L231 R cells. MRP gene amplification predominates, whereas rn the moderately resistant MOP& alls, geneamplification ISwmbmed with a mechanism nsulting in an additional increase in UK level of MRP mRNA. Fhtomsccnce in situ hybridiratron shows that, in the GLCU ADR cells. amplitied MRP xq!znces arc present both in double minute chromosomes (DM) and in homogeneously stainmg regions (HSR). By pulxd-field gel electrophoresis we show that the MRP-mntaining DM an I Mb in Icngtlt Chromosome-l6-specific repetitive sequences adjacent to the MRP gene arc also present in Ihe DM and HSR, campaliblc with the inwlvement of these sequences in recombination wcnts underlying h4lW gene amphtication. Our results show that low levels of drug resistance may arise by tmnxriptional activatmn of the MRP gene, whereas at high levels of drug resistance amplificalmn of the MRP gene predominates, posslbly facilitated by the prwence of recombination-prone sequences
Inhibition of proliferation by L-myc antisense DNA for the translational initiation site in human small cell lung cancer Dosaka-Akita H, Akie K. Hiroumi H. Kinoshita I. Kawakamr Y, Murakaml A. Fwsl Deparrnwnr of.WedKme. Hokkaido Unrversr~Sch of Bkdrcme. Norlh ,J, Wes, 7. .&to-ku. Sapporn 060. Cancer Res 1995;55 1559-64. We evaluated the antiprolifera0vc effect of L-myc antisense DNA m NCI- H209. a human small cell lung cancer (SCLC) cell line overexpressing the L- rnyc gene. The syntbetrc DNA used in the present study was oligodeoxynucleoside phospborothiaate, which showed rapid mcorpomtion into NC131209 cells and localized mainly m the cell nucleus and weakly in the cytoplasm. Tbc exposure of this cell line to L-mvc antisense DNA coverina the tnnslational initiation sile of Lmyc proteins inhibited the s& proliferation in a dose-dependent seauence-.sccciIic manner. Funhemmre. the mowh inhibitton by this antscn~ DNA was correlated with the level-of L- myc expression in three SCLC cell lines. NCI-H209, NCI-HS IO. and NCI-H82. In Western blot analysis, expression of the L-myc proteins was down-regulated in
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I
Increased prevalence of K-w oncogene mutations in lung adenocarcinoma Mills NE, Fishman CL. Ram WN, Dubin N. Jacobson DR. Research Service IJI. New York VA. Hosmlal, 423 E. 23 St.. New York, NY 10010. Cancer Res l995;55 1444-7. Reported estimates of ras mutation prevalence in lung adenocarcinomaof l5-24%maybcundexstimatesbecause oftbe insenatrnty of the assays used. We have devised a rapid, non-radioactive assay for ras mutations. which detects I mutant allcle/l0’ normal alleles and have used it to study DNA isolated from 53 lung tunwr samples (including 28 adenocarcinomas) previously analyzed by PCFUallele specific oligonucleotide hybridization, which is less sensitive. We detected m&ions in I3 of 28 samples, including 7 not detected by PCP/alkle so&tic oliaonucleotide hybndwation. We also found ras mutations in.14 of 25 ~wicusly unstudied samples (56%). Our resuits indicate that the prevalence of K-ras codon I2 mutations in lung adenocarciwma is higherthan previously reported; thus, ras mutations may be more clinically use&l as molecular markers for lung cancer than has been appreciated.
Neumlihromntosis type 2 (NF2) gene is somatically mutated in mesothelioma hut not in lung cancer Sekido Y. Pass HI. Bader S. Mew DN. Chnstman MF. Ca&r AF et al Smmons Cancer Centes Umrar.q v/ 7k,r SII Akdtcol (‘rr 5323 Horn Hrnes Bhd, Da//as. T . Y75235-8590 Cancer Rcs l’J95.55 1227. 31. WC have found 16 or28 small cell lung carlccrs. I7 or3 I non-llmill cell lung cancers. 2 of 3 carano~ds. and I2 of I4 mcsothclmmas thal had chromosome 22 cymgenetrc abnormalmes To dctcrmme whether the neurofibromalosis type 2 (NF2) gcnc lccalcd on chromosome 22 partic@es m the oncogencsls ofthcsc mahgnanacs. WC studxd DNAs from lung cancer cell lines and mesothclmmas usmg Southern blot analysis and the srnglc-strand conformauon polymorphism (SSCP) techmque for mutations covcrmg 8 of the 16 known NF2 ewns WC
Cancer 13 (1995) 81-104
Ca” rbd not a5ect the action potential production, whereas 5 iM TTX or subslttutton of Na* with cholineabolished rt. Action pMentiaIselicited rn the Ca”-free condition were reversibly blocked by 4 mM M&I, due to the Mn”-induced inhibition of voltage-dependent sodium currents (I(Na)) Therefore, Na’ channels, not Ca’* channels, underlie the excitability of SCLC cells. Whole-cell @a) was maximal with stepdepolarizing stimulations to 0 mV, and reversed at +45 2 mV, tn accord with the p=dicted Nemst ectuilibrium pofential for a Na+-selective channel. i(Na) evoked by dep&izing test potentials (-60 to +40 mV) exhibited a transient time course and activation/ mactivation kinetics typical of neuronal excitable membranes; the plot oftbe Hodgkin-Huxley parameters. m() and ho. also revealed biophysical similarity between SCLC and neumnal Na’ channels The single channel current amplitude, as nuasured with the inside-out patch co&guration. was I .O pi at -20 mV with a slope conductance of 12. I pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (L.ES), whtch are known to inhibit I(Ca) and I(Na) in bovine adrenal chmmaliin cells. also si8niticantly inhibited IlNa) in SCLC cells. These results indicate that (i)action po(endals in human SCLC cells nsult from the regenerative increase in voltage-gated Na* channel conductance: (ii) timdamental characteristics of SCLC Na’ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na’ channels are an additional target of the pathological IgG present in the patients’ sera
the armsense-treated cells compared tith control-treated cells in NCIH209. Togctkr with unique charaaeristia of the Llnyc gene. including: (a) a frequently amplified and overexpressed state in SCLC; and (b) very rcstictcd and low-level expression in human adult tissues. the present data indicate that L-myc is a good candidate for the target gene for antisenw DNA therapy based on molecular biological diagnosis in SCLC.
Mutations in the pl6(lNKJ)/MTSl/CDKN2, plS(INKdB)/ MTSZ, and pig genes in primary and metartatic lung cancer Okamoto A, Hussain SP, Hagiwara K, Spillare EA. Ruin MR. 1448-51. We exammed Ihe genomic status ofcychn&pmdent kinase-4 and -6 mlub~tors, pl6(INK4). pl5(INK4B). and ~18, I” 40 primary lung cancers and 31 metastatic lung cancers Alterations of the p16(INK4) gene were detected in 6 (2 wetirons and 4 homozygous deletions) of 22 metastat~ non-small cell lung cancers (NSCLCs: 27%). but none weredetected in 25 pnmay NSCLCs. 15 primarysmalloell lungcancers (SCLCs). or 9 metastatrc SCLCs. mdrcatin8 that mutation tn Ihe pl6(lNK4) gene IS a late event in NSCLC c&nogenesis Although three mtragenic mutationsofthc pIS(INK4B) gene were dctcctcd fin 25 prrmary NSCLCs (12%) and f,\e homozygous deletions of Ihe olS(INK4Bl eerie wcrc detected tn 22 NSCLCs 123%L no eenetx alteratiowofthepl5(INK4B) genewere foundin primary and metastauc SCLCs. The pl8 gene was wdd type tn these 7 I lung cancers. except I metastatic NSCLC whrch showed loss of hetcrwygowy We also esamincdaltcrat1onsofthcrthrcegennandespress~onofpl6(INK4) rn 21 human lung cancer cell hncs. Alteratmns of the plG(INK4) and plS(lNK4B) genes wcredetected I” 7l%ofthe NSCLC cell hnes (n = 14) and 50% of the NSCLC cell hncs (n = l4), rcspect~\cl>. but there were mane ,n the 7 SCLC cell hnes studwd No plR mutauons were dctcctedmthcsc21 cell hnes Thescraultr~nd~catethatbMhpl6lINK4~ andplS(INKJB)gene mutat,onsareasroc~rtcd~rth tumorprogrcsrmn ofa subset ofNSCLC. but not ofSCLC. and tbat plS(INK4B) mutatwns might also be an early event m the molecular pathogcncsrs of a subset or NSCLC .
Mechmims of MRP over-expression in four human lung-cancer cell lines and analysis of the MRP amplican Eiidems EWHM. De Haa M Ccco-Martin JM. Dttenheim CPE. Zaman &I. DawerseHG et al. &ision Uo/ecu/ar Biology Netherlands Cancer Insdtofe, Plesmanlaan 121, 1066 CYAmstenlam Int I Cancer l995;60:676-84. Some mtdtidrugresistant cell lines over-express Ihe gene encoding the multidrug-resistann-associaled protein f.MRP). In all all lines reported thus far, over-expression is assccialed with gene amplification. We have studied the predominant mechanisms of MRP over-expression in 4 human Iuwcanccr all lines that myer a range ofdntrrcsistance lewls, and we&e attalyzed the MRP ampli&. In th;SW-l573derived. waklv resistant cell line 30.3M. h4RP mRNA IS elevated 3fold in & &s&x of gene amplification. Run-on analysis shows that the increased MRP gene expression in this cell line is due lo transcriptional activation. In the highly resistant GLC4/ADR and CO&L231 R cells. MRP gene amplification predominates, whereas rn the moderately resistant MOP& alls, geneamplification ISwmbmed with a mechanism nsulting in an additional increase in UK level of MRP mRNA. Fhtomsccnce in situ hybridiratron shows that, in the GLCU ADR cells. amplitied MRP xq!znces arc present both in double minute chromosomes (DM) and in homogeneously stainmg regions (HSR). By pulxd-field gel electrophoresis we show that the MRP-mntaining DM an I Mb in Icngtlt Chromosome-l6-specific repetitive sequences adjacent to the MRP gene arc also present in Ihe DM and HSR, campaliblc with the inwlvement of these sequences in recombination wcnts underlying h4lW gene amphtication. Our results show that low levels of drug resistance may arise by tmnxriptional activatmn of the MRP gene, whereas at high levels of drug resistance amplificalmn of the MRP gene predominates, posslbly facilitated by the prwence of recombination-prone sequences
Inhibition of proliferation by L-myc antisense DNA for the translational initiation site in human small cell lung cancer Dosaka-Akita H, Akie K. Hiroumi H. Kinoshita I. Kawakamr Y, Murakaml A. Fwsl Deparrnwnr of.WedKme. Hokkaido Unrversr~Sch of Bkdrcme. Norlh ,J, Wes, 7. .&to-ku. Sapporn 060. Cancer Res 1995;55 1559-64. We evaluated the antiprolifera0vc effect of L-myc antisense DNA m NCI- H209. a human small cell lung cancer (SCLC) cell line overexpressing the L- rnyc gene. The syntbetrc DNA used in the present study was oligodeoxynucleoside phospborothiaate, which showed rapid mcorpomtion into NC131209 cells and localized mainly m the cell nucleus and weakly in the cytoplasm. Tbc exposure of this cell line to L-mvc antisense DNA coverina the tnnslational initiation sile of Lmyc proteins inhibited the s& proliferation in a dose-dependent seauence-.sccciIic manner. Funhemmre. the mowh inhibitton by this antscn~ DNA was correlated with the level-of L- myc expression in three SCLC cell lines. NCI-H209, NCI-HS IO. and NCI-H82. In Western blot analysis, expression of the L-myc proteins was down-regulated in
.
_I
I
Increased prevalence of K-w oncogene mutations in lung adenocarcinoma Mills NE, Fishman CL. Ram WN, Dubin N. Jacobson DR. Research Service IJI. New York VA. Hosmlal, 423 E. 23 St.. New York, NY 10010. Cancer Res l995;55 1444-7. Reported estimates of ras mutation prevalence in lung adenocarcinomaof l5-24%maybcundexstimatesbecause oftbe insenatrnty of the assays used. We have devised a rapid, non-radioactive assay for ras mutations. which detects I mutant allcle/l0’ normal alleles and have used it to study DNA isolated from 53 lung tunwr samples (including 28 adenocarcinomas) previously analyzed by PCFUallele specific oligonucleotide hybridization, which is less sensitive. We detected m&ions in I3 of 28 samples, including 7 not detected by PCP/alkle so&tic oliaonucleotide hybndwation. We also found ras mutations in.14 of 25 ~wicusly unstudied samples (56%). Our resuits indicate that the prevalence of K-ras codon I2 mutations in lung adenocarciwma is higherthan previously reported; thus, ras mutations may be more clinically use&l as molecular markers for lung cancer than has been appreciated.
Neumlihromntosis type 2 (NF2) gene is somatically mutated in mesothelioma hut not in lung cancer Sekido Y. Pass HI. Bader S. Mew DN. Chnstman MF. Ca&r AF et al Smmons Cancer Centes Umrar.q v/ 7k,r SII Akdtcol (‘rr 5323 Horn Hrnes Bhd, Da//as. T . Y75235-8590 Cancer Rcs l’J95.55 1227. 31. WC have found 16 or28 small cell lung carlccrs. I7 or3 I non-llmill cell lung cancers. 2 of 3 carano~ds. and I2 of I4 mcsothclmmas thal had chromosome 22 cymgenetrc abnormalmes To dctcrmme whether the neurofibromalosis type 2 (NF2) gcnc lccalcd on chromosome 22 partic@es m the oncogencsls ofthcsc mahgnanacs. WC studxd DNAs from lung cancer cell lines and mesothclmmas usmg Southern blot analysis and the srnglc-strand conformauon polymorphism (SSCP) techmque for mutations covcrmg 8 of the 16 known NF2 ewns WC