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Development of a broad spectrum PCR assay for papillomaviruses and its application in screening lung cancer biopsies
428
Abstracts / Lung Cancer I I (1994) 423-444
cancer cell lines. In contrast, IA-l mRNA was detected in only 13 % (4 of 30) of non-small cell lung cancer cell lines. Nine of the 30 (30%) expressed either chromogranin A mRNA or produced L-dopa decarboxylase. Four of these 9 (44%) had detectable levels of IA-1 mRNA. In most of the lung cancer cell lines examined, IA-1 showed high concordance with the other neuroendocrine markers, L-dopa decarboxylase, and chromogranin A. The one exception was a variant small cell lung cancer cell line which expressed low or nondetectable levels of Ldopa decarboxylase. IA-1 is a candidate marker of neuroendocrine differentiation of human lung tumors.
A retroriral wild-type pS3 expression rector penetratg human lung cancer spheroids and inhibits growth by inducing apoptosis Fujiwara T, Grimm EA. Mukhopadhyay T, De Wei Cai Owen-Schaub LB, Roth JA. lhoracic/Cardiovascular Surg. Dept., University of Teras, M. D. Am&son Cancer Center, Houston, Tw 77030. Cancer Res 1993;53:4129-33. Multicellular tumor spheroids approximate the three-dimensional configurationofprimatyand metastatictumors. Theeffectsofretrovimsmediated transduction of wild-type p53 (wt-~53) were studied on multicellular tumor spheroids of human non-small cell lung cancer cell lines H322.a, the p53 gene of which is homoxygously mutated at codon 248, and WT2266, which has endogenous wt-~53. The growth of WT226b spheroids was not affected by exogenous wt-~53 transduction; the growth of H322a spheroids, however, was significantly inhibited by the addition of w&p53 virus stocks. Transduction of cells by the wtp53 retroviral vector and penetration of multiple cell layers in H322a spheroids was demonstrated by in situ polymerase chain reaction/ hybridization with theneomycin-resistantneoprobe. Apoptoticchanges indicating programmedcell death were observed in H322a spheroids treated with the wt-~53 virus. These results suggest that retroviral vectors can penetrate into multiple cell layers of three-dimensional tumor masses and induce potentially therapeutic effects.
Development of a broad spectrum PCR asspy for papillomaviruses and its application in screening lung cancer biopsies Shamanin V, Delius H, De Villiers EM. Div TumourvirusCharacterisation. Deursches Krebsforschungsz.enhwn. Im Neuenheimer Feld 242, 69120 Heidelberg. J Gen Viral 1994;lS: 1149-56. A PCR assay was developed to detect known and as yet unidentified papillomaviruses (PVs). For this purpose we analysed the conserved amino acid sequences in the Ll and E 1 open reading frames of 45 human and nine animal PVs. Candidate regions for the design of a primer were identified as three having the least number of amino acid and nucleotide sequencevariantsamongthedifferent PVs. TheseregionsintheLl ORF have been described previously. We modified the sequences of the backward and the forward primers, as well as the sequence of the oligonucleotide used as the degenerate probe,in order to cover a broader spectrum of PVs. The sensitivity of the assay for the human and animal PVs tested after hybridization with a “P-labelled degenerate oligonucleotide probe was one genome copy per cell for integrated PV DNA and 10 genome copies per cell for plasmid PV DNA. The ooly exceptions were human papillomavirus (HPV) type 4, HPV60 and HPV65, for which a lower sensitivity was obtained. This group could be detected only by using additional primers. The assay was used to analyse 85 lung cancer biopsies representing different histological types. Using this system no PV DNA sequences were detected in the biopsies when compared with human placental DNA (a negative control) and PV DNA-positive standards.
Homozygous deletion on chromosome 9p and loss of heterozygosity on 9q, 6p, and 6q in primary human small cell long cancer Merlo A, Gabrielson E, Mabry M, Vollmer R, Baylin SB, Sidransky D. Div. of Head/Neck Cancer Research, Depanment of Otohnyngology, Johns Hopkins School of Medicine, 720 Rurland Avenue, Baliimore, MD 21205. Cancer Res 1994;54:2322-6. Weanalyzed the pattern of allelic loss in 33 primary human small cell lung cancers (SCLCs) using highly informative microsatellite markers on chromosomes 2p, 3p, 5q, 6, 9, 13q, and 17~. Nineteen of these tumors (58%) displayed loss of heteroxygosity on chromosome 9. Fourteen SCLCs demonstrated loss of heteroxygosity for all informative markers on both chromosomal arms; two tumors demonstrated partial loss on chromosome 9p. In one tumor, a multiplex polymerase chain reaction assay disclosed a homoxygous deletion at 9p2 l-22 including the markers IFN-a, D9Sl26, and D9Sl71. Two SCLCs retained all informative markers on 9p but showed allelic loss of the entire 9q arm, while one case had a partial loss of proximal 9q extending into all of 9p. Analysis of other chromosomal arms showed loss of heteroxygosity on 3p(93%),5q(75%),6p(46%),6q(47%),13q(75%),andl7p(93%). It was necessary to test multiple markers at several loci because of the frequent expression of microsatellite instability that confounded our mappingeffortsinSCLCswithteplicationerrors.Thisstudydemonstratea the frequent loss ofa suppressor gene locus on chromosome 9p2 1-22 and identifies novel suppressor loci on 6p, 6q, and 9q in primary SCLC.
Genetic instability of microsatellite sequences in many non-small cell lung carcinomas Shridhar V, Siegfried J, HuntJ, Del Mar Alonso M, Smith DI. Division of Hematology/Oncology, Depamnenr of Internal Medicine, Wayne State Univ. School of Medicine, 540 East Canfield. Detroir. MI 48201. Cancer Res 1994 5418 (2084-2087) We have analyzed DNA obtained from 38 lung tumors and normal lung or blood DNA for microsatellite instability. Instability was examined at 10 different microsatellite loci on chromosome 3p, as well as loci on 3q, llp, llq, and 13q, and two on Xq. We observed microsatellite instability at one or more loci in 13 of the lung tumors analyzed, and this instability ranged from tumors showing instability in only a single microsatellite to two adenocarcinomas that had alterations in all 16 tested microsatellites. Microsatellite instability could therefore play a significant role in the development of a sizable portion of lung tumors.
A J-Mb physical map of the chromosome region 8p21.3-~22, including a 600- kb regioncommonly deleted in human hepatocelhdar carcinoma, coIorectal cancer, and non-small cell lung cancer Fujiwars Y, Ohata H, Emi M, Okui K. Koyama K, Tsuchiya E et al. DepartmentofBiochemistrystry.Cancerlnstitute, l-37-1, Kami-ikebukuro, Toshima-ku, Tokyo I70. Genes Chromosomes Cancer 1994;10:7-14. To isolate a putative tumor suppressor gene(s), we have constructed a physical map and a detailed deletion map of chromosome region 8p21.3-~22, where loss of heterozygosity (LOH) has been frequently seen in human hepatocelhdar carcinomas (HCC), color&al cancers (CRC), and non-small cell lung cancers (NSCLC). The smallest commonly deleted region at 8p21.3-p22 in HCC and CRC was between the loci defined by C18-245 and Cl8-2644; in NSCLC, a region between Cl8-1051 and Cl8-2644 was commonly deleted. Acontiguous physical map of 12 cosmid markers in the 8p21.3-p22 region was constructed by means of multi-color fluorescence in situ hybridization (FISH) and pulsed- field gel electrophoresis (PFGE). On the basis of this physical map, which spans roughly 3.1 Mb, the estimated sixes of the
Abstracts / Lung Cancer I I (1994) 423-444
cancer cell lines. In contrast, IA-l mRNA was detected in only 13 % (4 of 30) of non-small cell lung cancer cell lines. Nine of the 30 (30%) expressed either chromogranin A mRNA or produced L-dopa decarboxylase. Four of these 9 (44%) had detectable levels of IA-1 mRNA. In most of the lung cancer cell lines examined, IA-1 showed high concordance with the other neuroendocrine markers, L-dopa decarboxylase, and chromogranin A. The one exception was a variant small cell lung cancer cell line which expressed low or nondetectable levels of Ldopa decarboxylase. IA-1 is a candidate marker of neuroendocrine differentiation of human lung tumors.
A retroriral wild-type pS3 expression rector penetratg human lung cancer spheroids and inhibits growth by inducing apoptosis Fujiwara T, Grimm EA. Mukhopadhyay T, De Wei Cai Owen-Schaub LB, Roth JA. lhoracic/Cardiovascular Surg. Dept., University of Teras, M. D. Am&son Cancer Center, Houston, Tw 77030. Cancer Res 1993;53:4129-33. Multicellular tumor spheroids approximate the three-dimensional configurationofprimatyand metastatictumors. Theeffectsofretrovimsmediated transduction of wild-type p53 (wt-~53) were studied on multicellular tumor spheroids of human non-small cell lung cancer cell lines H322.a, the p53 gene of which is homoxygously mutated at codon 248, and WT2266, which has endogenous wt-~53. The growth of WT226b spheroids was not affected by exogenous wt-~53 transduction; the growth of H322a spheroids, however, was significantly inhibited by the addition of w&p53 virus stocks. Transduction of cells by the wtp53 retroviral vector and penetration of multiple cell layers in H322a spheroids was demonstrated by in situ polymerase chain reaction/ hybridization with theneomycin-resistantneoprobe. Apoptoticchanges indicating programmedcell death were observed in H322a spheroids treated with the wt-~53 virus. These results suggest that retroviral vectors can penetrate into multiple cell layers of three-dimensional tumor masses and induce potentially therapeutic effects.
Development of a broad spectrum PCR asspy for papillomaviruses and its application in screening lung cancer biopsies Shamanin V, Delius H, De Villiers EM. Div TumourvirusCharacterisation. Deursches Krebsforschungsz.enhwn. Im Neuenheimer Feld 242, 69120 Heidelberg. J Gen Viral 1994;lS: 1149-56. A PCR assay was developed to detect known and as yet unidentified papillomaviruses (PVs). For this purpose we analysed the conserved amino acid sequences in the Ll and E 1 open reading frames of 45 human and nine animal PVs. Candidate regions for the design of a primer were identified as three having the least number of amino acid and nucleotide sequencevariantsamongthedifferent PVs. TheseregionsintheLl ORF have been described previously. We modified the sequences of the backward and the forward primers, as well as the sequence of the oligonucleotide used as the degenerate probe,in order to cover a broader spectrum of PVs. The sensitivity of the assay for the human and animal PVs tested after hybridization with a “P-labelled degenerate oligonucleotide probe was one genome copy per cell for integrated PV DNA and 10 genome copies per cell for plasmid PV DNA. The ooly exceptions were human papillomavirus (HPV) type 4, HPV60 and HPV65, for which a lower sensitivity was obtained. This group could be detected only by using additional primers. The assay was used to analyse 85 lung cancer biopsies representing different histological types. Using this system no PV DNA sequences were detected in the biopsies when compared with human placental DNA (a negative control) and PV DNA-positive standards.
Homozygous deletion on chromosome 9p and loss of heterozygosity on 9q, 6p, and 6q in primary human small cell long cancer Merlo A, Gabrielson E, Mabry M, Vollmer R, Baylin SB, Sidransky D. Div. of Head/Neck Cancer Research, Depanment of Otohnyngology, Johns Hopkins School of Medicine, 720 Rurland Avenue, Baliimore, MD 21205. Cancer Res 1994;54:2322-6. Weanalyzed the pattern of allelic loss in 33 primary human small cell lung cancers (SCLCs) using highly informative microsatellite markers on chromosomes 2p, 3p, 5q, 6, 9, 13q, and 17~. Nineteen of these tumors (58%) displayed loss of heteroxygosity on chromosome 9. Fourteen SCLCs demonstrated loss of heteroxygosity for all informative markers on both chromosomal arms; two tumors demonstrated partial loss on chromosome 9p. In one tumor, a multiplex polymerase chain reaction assay disclosed a homoxygous deletion at 9p2 l-22 including the markers IFN-a, D9Sl26, and D9Sl71. Two SCLCs retained all informative markers on 9p but showed allelic loss of the entire 9q arm, while one case had a partial loss of proximal 9q extending into all of 9p. Analysis of other chromosomal arms showed loss of heteroxygosity on 3p(93%),5q(75%),6p(46%),6q(47%),13q(75%),andl7p(93%). It was necessary to test multiple markers at several loci because of the frequent expression of microsatellite instability that confounded our mappingeffortsinSCLCswithteplicationerrors.Thisstudydemonstratea the frequent loss ofa suppressor gene locus on chromosome 9p2 1-22 and identifies novel suppressor loci on 6p, 6q, and 9q in primary SCLC.
Genetic instability of microsatellite sequences in many non-small cell lung carcinomas Shridhar V, Siegfried J, HuntJ, Del Mar Alonso M, Smith DI. Division of Hematology/Oncology, Depamnenr of Internal Medicine, Wayne State Univ. School of Medicine, 540 East Canfield. Detroir. MI 48201. Cancer Res 1994 5418 (2084-2087) We have analyzed DNA obtained from 38 lung tumors and normal lung or blood DNA for microsatellite instability. Instability was examined at 10 different microsatellite loci on chromosome 3p, as well as loci on 3q, llp, llq, and 13q, and two on Xq. We observed microsatellite instability at one or more loci in 13 of the lung tumors analyzed, and this instability ranged from tumors showing instability in only a single microsatellite to two adenocarcinomas that had alterations in all 16 tested microsatellites. Microsatellite instability could therefore play a significant role in the development of a sizable portion of lung tumors.
A J-Mb physical map of the chromosome region 8p21.3-~22, including a 600- kb regioncommonly deleted in human hepatocelhdar carcinoma, coIorectal cancer, and non-small cell lung cancer Fujiwars Y, Ohata H, Emi M, Okui K. Koyama K, Tsuchiya E et al. DepartmentofBiochemistrystry.Cancerlnstitute, l-37-1, Kami-ikebukuro, Toshima-ku, Tokyo I70. Genes Chromosomes Cancer 1994;10:7-14. To isolate a putative tumor suppressor gene(s), we have constructed a physical map and a detailed deletion map of chromosome region 8p21.3-~22, where loss of heterozygosity (LOH) has been frequently seen in human hepatocelhdar carcinomas (HCC), color&al cancers (CRC), and non-small cell lung cancers (NSCLC). The smallest commonly deleted region at 8p21.3-p22 in HCC and CRC was between the loci defined by C18-245 and Cl8-2644; in NSCLC, a region between Cl8-1051 and Cl8-2644 was commonly deleted. Acontiguous physical map of 12 cosmid markers in the 8p21.3-p22 region was constructed by means of multi-color fluorescence in situ hybridization (FISH) and pulsed- field gel electrophoresis (PFGE). On the basis of this physical map, which spans roughly 3.1 Mb, the estimated sixes of the