Detection of amplified genomic sequences in human small-cell lung carcinoma cells by arbitrarily primed-PCR genomic fingerprinting

The arbitrarily primed-PCR (AP-PCR) genomic fingerprinting method was applied to evaluate its effectiveness in detecting and characterizing amplified DNA fragments in two small-cell lung carcinoma (SCLC) cell lines, NCI-H69 and NCI-H82. Of the 2428 DNA fragments detected by AP-PCR using 62 arbitrary primers, 2 (0.08%) DNA fragments were amplified in NCI-H69 and 6 (0.25%) DNA fragments were amplified in NCI-H82. Based on these results, we estimate the total size of the amplified genomic regions in these cell lines to be 3000 megabase pairs (Mb) × 0.0008 = 2.4 Mb in NCI-H69 and 3000 Mb × 0.0025 = 7.5 Mb in NCI-H82. The 2 amplified fragments in NCI-H69 were mapped to chromosome 2, and all 6 amplified fragments in NCI-H82 were mapped to chromosome 8. This strongly suggests that restricted chromosomal regions are specifically amplified in these SCLC cell lines. Since the N-myc gene at 2p24 is amplified in NCI-H69 and the c-myc gene at 8q24 is amplified in NCI-H82, it is possible that these DNA fragments are co-amplified with N-myc or c-myc in these cell lines. However, since the 2 amplified fragments in NCI-H69 were not amplified in 42 other human cancer cell lines including 11 cell lines carrying amplified N-myc genes, it is also possible that there are amplified regions on chromosome 2 other than the N-myc locus at 2p24 in NCI-H69. In contrast, all 6 amplified fragments in NCI-H82 were amplified in several other human cancer cell lines carrying amplified c-myc genes. This result further indicates that these fragments were derived from an amplification unit that includes the c-myc gene. Our results show the ability of the AP-PCR method to analyze the fraction of the genome with amplification in human cancer cells. Received: 10 April 1995 / Revised: 18 December 1995, 15 April 1996     

Differential expression of adrenomedullin and its receptor component, receptor activity modifying protein (RAMP) 2 during hypoxia in cultured human neuroblastoma cells.

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex consisting of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 2. To explore possible pathophysiological roles of adrenomedullin and its receptor component RAMP2 in hypoxic tissues, we studied effects of hypoxia on expression of adrenomedullin and RAMP2 in two human neuroblastoma cell lines, IMR-32 and NB69, by radioimmunoassay and Northern blot analysis. Expression levels of adrenomedullin were increased by hypoxia in both cell lines. Treatment with cobalt chloride or desferrioxamine mesylate also increased expression levels of adrenomedullin mRNA. On the other hand, expression levels of RAMP2 mRNA were decreased in IMR-32 cells and were not changed in NB69 cells by hypoxia. Treatment with cobalt chloride or desferrioxamine mesylate decreased expression levels of RAMP2 mRNA in both IMR-32 and NB69 cells. These findings indicate that adrenomedullin expression is induced during hypoxia in IMR-32 and NB69 neuroblastoma cells, but RAMP2 expression is rather suppressed under the same conditions. The decreased expression of RAMP2 and the ADM expression induction under hypoxia may constitute one mechanism of cellular adaptation to hypoxic stress.     

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification.     

A new benzophenone from Mesua congestiflora,an inhibitor against human B lymphocyte cancer cell line

An investigation on the secondary metabolites from the roots of Mesua congestiflora was carried out. A new benzophenone–congestiflorone (1) and one xanthone, α-mangostin (2) were obtained. Structures of these compounds were determined using spectroscopic methods which included 1D and 2D NMR, GC–MS, IR and UV techniques. The cytotoxic activities of this new metabolite against cancer cell lines such as SNU-1, LS-174T, HeLa, SK-MEL-28, NCI-H23, IMR-32, Hep-G2 and K562 were evaluated using the MTT assays. Congestiflorone (1) gave good cytotoxic activity against Raji lymphoma cell line.     

蓖麻根提取物对HepG2,NCI-H460和SGC-7901细胞增殖及凋亡作用的影响

探讨蓖麻根不同提取物对肝癌HepG2细胞株、肺癌NCI-H460细胞株和胃癌SGC-7901细胞株增殖及其凋亡的影响.采用MTT法检测蓖麻根不同提取物处理48h、72h对HepG2细胞、NCI-H460细胞和SGC-7901细胞增殖的抑制率;Hoechst 33258荧光染料染色法观察HepG2细胞凋亡,流式细胞术检测...     

The human homolog of the rodent immediate early response genes, PC4 and TIS7, resides in the lung cancer tumor suppressor gene region on chromosome 3p21

Recently, human chromosome band 3p21.3 was shown to undergo overlapping homozygous deletions in several small cell lung cancer lines further defining a putative tumor suppressor gene(s) region. We report the cloning and mutational analysis of a novel human gene, SKMc15, from the commonly homozygously deleted region in three small cell lung cancer lines (NCI-H1450, NCI-H740, GLC20). It has 11 exons ranging in size from 50 to 541 bp with an open reading frame of 442 amino acids. The gene covers 7 to 10 kb of genomic DNA; the message of 1.8 to 2 kb is expressed in all analyzed fetal and adult human and mouse tissues including heart, brain, placenta, lung liver, skeletal muscle, kidney, testis and pancreas and in small cell and non-small cell cancer lines. The intron/exon boundaries were used to analyze the gene for mutations by exon PCR-SSCP sequencing in 60 small cell lung cancer cell lines. No loss-of-function mutations were detected. The cDNA sequence has high homology, 75% at the protein level, to the rat early response gene PC4 and its murine homolog TIS7. In addition, the known partial sequence of the putative mouse interferon β2 (64 amino acids) gene is highly conserved in PC4/TIS7 (94%) and in SKMc15 (83%) at the amino acid level. The sequence TAAAT, which is thought to be involved in mRNA degradation, is present in the 3′ UTR of SKMc15 and in the 3′ UTR of PC4 and TIS7 genes. Received: 28 August 1996 / Revised: 18 October 1996     

Correlating breakage-fusion-bridge events with the overall chromosomal instability and in vitro karyotype evolution in prostate cancer

Chromosomal instability (CIN) is thought to underlie the generation of chromosomal changes and genomic heterogeneity during prostatic tumorigenesis. The breakage-fusion-bridge (BFB) cycle is one of the CIN mechanisms responsible for characteristic mitotic abnormalities and the occurrence of specific classes of genomic rearrangements. However, there is little detailed information concerning the role of BFB and CIN in generating genomic diversity in prostate cancer. In this study we have used molecular cytogenetic methods and array comparative genomic hybridization analysis (aCGH) of DU145, PC3, LNCaP, 1532T and 1542T to investigate the in vitro role of BFB as a CIN mechanism in karyotype evolution. Analysis of mitotic structures in all five prostate cancer cell lines showed increased frequency of anaphase bridges and nuclear strings. Structurally rearranged dicentric chromosomes were observed in all of the investigated cell lines, and Spectral Karyotyping (SKY) analysis was used to identify the participating rearranged chromosomes. Multicolor banding (mBAND) and aCGH analysis of some of the more complex chromosomal rearrangements and associated amplicons identified inverted duplications, most frequently involving chromosome 8. Chromosomal breakpoint analysis showed there was a higher frequency of rearrangement at centromeric and pericentromeric genomic regions. The distribution of inverted duplications and ladder-like amplifications was mapped by mBAND and by aCGH. Adjacent spacing of focal amplifications and microdeletions were observed, and focal amplification of centromeric and end sequences was present, particularly in the most unstable line DU145. SKY analysis of this line identified chromosome segments fusing with multiple recipient chromosomes (jumping translocations) identifying potential dicentric sources. Telomere free end analysis indicated loss of DNA sequence. Moreover, the cell lines with the shortest telomeres had the most complex karyotypes, suggesting that despite the expression of telomerase, the reduced telomere length could be driving the observed BFB events and elevated levels of CIN in these lines.     

Label-Free Quantitative Proteomics and N-terminal Analysis of Human Metastatic Lung Cancer Cells

Proteomic analysis is helpful in identifying cancer-associated proteins that are differentially expressed and fragmented that can be annotated as dysregulated networks and pathways during metastasis. To examine meta-static process in lung cancer, we performed a proteomics study by label-free quantitative analysis and N-terminal analysis in 2 human non-small-cell lung cancer cell lines with disparate metastatic potentials—NCI-H1703 (primary cell, stage I) and NCI-H1755 (metastatic cell, stage IV). We identified 2130 proteins, 1355 of which were common to both cell lines. In the label-free quantitative analysis, we used the NSAF normalization method, resulting in 242 differential expressed proteins. For the N-terminal proteome analysis, 325 N-terminal peptides, including 45 novel fragments, were identified in the 2 cell lines. Based on two proteomic analysis, 11 quantitatively expressed proteins and 8 N-terminal peptides were enriched for the focal adhesion pathway. Most proteins from the quantitative analysis were upregulated in metastatic cancer cells, whereas novel fragment of CRKL was detected only in primary cancer cells. This study increases our understanding of the NSCLC metastasis proteome.     

Molecular cytogenetic characterization of the 11q13 amplicon in head and neck squamous cell carcinoma

Amplification of 11q13 DNA sequences and overexpression of CCND1 are common findings in head and neck squamous cell carcinoma (HNSCC), identified in about 30% of the cases. However, little is known about initiation of the amplification and the organization of the amplicon. In order to study the structure of the amplicon in more detail and to learn more about the mechanisms involved in its initiation, prometaphase, metaphase, and anaphase fluorescence in situ hybridization (FISH) with 40 BAC clones spanning a 16-Mb region in chromosome bands 11q12.2 to 11q13.5 was performed in nine HNSCC cell lines with homogeneously staining regions. FISH analysis showed that the size of the amplicon varied among the nine cell lines, the smallest being 2.12 Mb and the largest 8.97 Mb. The smallest overlapping region of amplification was approximately 1.61 Mb, covering the region from BAC 729E14 to BAC 102B19. This region contained several genes previously shown to be amplified and overexpressed in HNSCC, including CCDN1, CTTN, SHANK2, and ORAOV1. The cell lines were also used to study the internal structure of the amplicon. Various patterns of amplified DNA sequences within the amplicon were found among the nine cell lines. Even within the same cell line, different amplicon structures could be found in different cell populations, indicating that the mechanisms involved in the development of the amplicons in HNSCC were more complex than previously assumed. The frequent finding of inverted repeats within the amplicons, however, suggests that breakage-fusion-bridge cycles are important in the initiation, but the fact that such repeats constituted only small parts of the amplicons indicate that they are further rearranged during tumor progression.     

Integrated cytogenetic and high-resolution array CGH analysis of genomic alterations associated with MYCN amplification

Amplification of oncogenes and closely linked flanking genes is common in some types of cancer and can be associated with complex chromosome rearrangements and/or co-amplification of non-syntenic chromosomal regions. To better understand the etiology and structural complexity of focal MYCN amplicons in human neuronal cancer, we investigated the precise chromosomal locations of high copy number genomic regions in MYCN amplified cell lines. An integrated cytogenetic map of the MYCN amplicon was created using high-resolution array CGH, spectral karyotyping (SKY), multi-color banding (mBAND), and fluorescence in situ hybridization (FISH) in 4 human neuronal tumor cell lines. The evidence of complex intra- and inter-chromosomal events, providing clues concerning the nature of the genomic mechanisms that contributed to the process of MYCN amplification, was observed. The presence of multiple co-amplified syntenic or non-syntenic sequences in the MYCN amplicon is quite intriguing. MYCN is usually centrally located in the amplicon; however, the structure and complexity of the amplicons were highly variable. It is noteworthy that clusters of unstable repetitive regions characterized by CNV sequences were present throughout the regions encompassed by MYCN gene amplification, and these sequences could provide a mechanism to destabilize this region of the genome. Complex structural rearrangements involving genomic losses and gains in the 2p24 region lead to MYCN amplification and that these rearrangements can trigger amplification events.     

High affinity binding of cholecystokinin to small cell lung cancer cells

The binding of 125I-Bolton Hunter-cholecystokinin octapeptide (125I-BH-CCK-8) to small cell lung cancer cell lines was investigated. 125I-BH-CCK-8 bound with high affinity (Kd = 2.4 nM) to an apparent single class of sites (1700/cell) using cell line NCI-H209. Binding was time dependent and the ratio of specific/nonspecific binding was 8/1. Pharmacology studies indicated that gastrin, caerulein, CCK-33 and nonsulfated CCK-8 were potent inhibitors of specific 125I-BH-CCK-8 binding whereas CCK-26-32-NH2 was not. Because CCK receptors are present on small cell lung cancer cells, CCK may function as a regulatory peptide in this disease.     

3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.     

Comparative chromosome analysis of nine squamous cell carcinoma lines from tumors of the head and neck

The chromosomes of nine cell lines from human squamous cell carcinomas of the head and neck were examined. Five of the lines were derived from patients who had received chemotherapy and radiotherapy, one line was derived from a lymph-node metastasis from one of these patients, and three lines were from patients who had not been treated. The cell lines from the untreated patients had lower modal chromosome numbers than those of all but one of the lines from the treated patients. A structural abnormality involving chromosome 1 with breakpoints located in p13, p22, q21, or q32 was found in all cases. The NRAS and SK oncogenes are located in or near some of these segments. In four of the cell lines the nucleolus organizer regions (NORs) were 10-30 times larger than the NORs of the normal human chromosomes. Double minutes (DM) and homogeneously staining regions (HSR) were found in four of the lines.     

Apparent noncompetitive antagonism of muscarinic receptor mediated Ca2+ mobilization by some muscarinic antagonists.

Ca2+ mobilizations in SH-SY5Y and IMR-32 human neuroblastoma cell lines were measured using the fluorescent Ca2+ indicator fura-2. A variety of antagonists (atropine, pirenzepine, 4-DAMP and N-methyl-scopolamine) inhibited carbamyl choline-induced transient Ca2+ mobilization both in a competitive and a noncompetitive manner. The apparent noncompetitive inhibition constants were lower in IMR-32 than in SH-SY5Y cells even when the competitive inhibition constants were similar. This may relate to the previously reported differential expression of muscarinic receptor subtypes in these cell lines.     

Establishment of an immunoscreening system using recombinant VP1 protein for the isolation of a monoclonal antibody that blocks JC virus infection

Polyomavirus JC (JCV) infection causes the fatal human demyelinating disease, progressive multifocal leukoencephalopathy. Although the initial interaction of JCV with host cells occurs through direct binding of the major viral capsid protein (VP1) with cell-surface molecules possessing sialic acid, these molecules have not yet been identified. In order to isolate monoclonal antibodies which inhibit attachment of JCV, we established an immunoscreening system using virus-like particles consisting of the VP1. Using this system, among monoclonal antibodies against the cell membrane fraction from JCV-permissive human neuroblastoma IMR-32 cells, we isolated a monoclonal antibody designated as 24D2 that specifically inhibited attachment and infection of JCV to IMR-32 cells. The antibody 24D2 recognized a single molecule of around 60 kDa in molecular weight in the IMR-32 membrane fraction. Immunohistochemical staining with 24D2 demonstrated immunoreactivity in the cell membrane of JCV-permissive cell lines and glial cells of the human brain. These results suggested that the molecule recognized by 24D2 plays a role in JCV infection, and that it might participate as a receptor or a co-receptor in JCV attachment and entry into the cells.