Expression of the Elm1 gene, a novel gene of the CCN (connective tissue growth factor, Cyr61/Cef10, and neuroblastoma overexpressed gene) family, suppresses In vivo tumor growth and metastasis of K-1735 murine melanoma cells.

We previously isolated a partial cDNA fragment of a novel gene, Elm1 (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells. Here we determined the full-length cDNA structure of Elm1 and investigated the effect of Elm1 expression on growth and metastatic potential of K-1735 cells. The Elm1 gene encodes a predicted protein of 367 amino acids showing approximately 40% amino acid identity with the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, neuroblastoma overexpressed gene [Nov]) family proteins, which consist of secreted cysteine-rich proteins with growth regulatory functions. Elm1 is also a cysteine-rich protein and contains a signal peptide and four domains conserved in the CCN family proteins. Elm1 was highly conserved, expressed ubiquitously in diverse organs, and mapped to mouse chromosome 15. High-metastatic K-1735 M-2 cells, which did not express Elm1, were transfected with an Elm1 expression vector, and several stable clones with Elm1 expression were established. The in vivo growth rates of cells expressing a high level of Elm1 were remarkably slower than those of cells expressing a low level of Elm1. Metastatic potential of transfectants was reduced in proportion to the level of Elm1 expression. Thus, Elm1 is a novel gene of CCN family that can suppress the in vivo growth and metastatic potential of K-1735 mouse melanoma cells.

Alterations of myc, myb, and rasHa proto-oncogenes in cancers are frequent and show clinical correlation.

Alterations of c-myc, c-rasHa, or c-myb oncogenes were found in more than one-third of human solid tumors. Amplification of c-myc occurred in advanced, widespread tumors or in aggressive primary tumors. Apparent allelic deletions of c-rasHa and c-myb can be correlated with progression and metastasis of carcinomas and sarcomas.

Prevalence and specificity of LKB1 genetic alterations in lung cancers.

Germline LKB1 mutations cause Peutz-Jeghers syndrome, a hereditary disorder that predisposes to gastrointestinal hamartomatous polyposis and several types of malignant tumors. Somatic LKB1 alterations are rare in sporadic cancers, however, a few reports showed the presence of somatic alterations in a considerable fraction of lung cancers. To determine the prevalence and the specificity of LKB1 alterations in lung cancers, we examined a large number of lung cancer cell lines and lung adenocarcinoma (AdC) specimens for the alterations. LKB1 genetic alterations were frequently detected in the cell lines (21/70, 30%), especially in non-small cell lung cancers (NSCLCs) (20/51, 39%), and were significantly more frequent in cell lines with KRAS mutations. Point mutations were detected only in AdCs and large cell carcinomas, whereas homozygous deletions were detected in all histological types of lung cancer. Among lung AdC specimens, LKB1 mutations were found in seven (8%) of 91 male smokers but in none of 64 females and/or nonsmokers, and were significantly more frequent in poorly differentiated tumors. The difference in the frequency of LKB1 alterations between cell lines and tumor specimens was likely to be owing to masking of deletions by the contamination of noncancerous cells in the tumor specimens. These results indicate that somatic LKB1 genetic alterations preferentially occur in a subset of poorly differentiated lung AdCs that appear to correlate with smoking males.

Identification of tumor markers and differentiation markers for molecular diagnosis of lung adenocarcinoma.

To identify tumor markers and differentiation markers for lung adenocarcinoma (AdC), we analysed expression profiles of 14,500 genes against three cases of type II alveolar epithelial cells, bronchiolar epithelial cells, and bronchial epithelial cells, respectively, and 10 cases of AdC cells isolated by laser capture microdissection. Hierarchical clustering analysis indicated that AdC cells and noncancerous lung epithelial cells are significantly different in their expression profiles, and that different sets of differentiation markers are expressed among alveolar, bronchiolar and bronchial epithelial cells. Nine genes were identified as being highly expressed in AdC cells, but not expressed in noncancerous lung epithelial cells. Sixteen genes were identified as differentiation markers for lung epithelial cells. Real-time RT-PCR analysis of 45 lung AdC cases further revealed that expression of four tumor markers in AdC cells was significantly higher than that in noncancerous lung cells and that expression of ten differentiation markers was retained in a considerable fraction of lung AdC cases. Five tumor markers and seven differentiation markers were not expressed in peripheral blood cells. Similarities and differences in expression profiles between normal epithelial cells from different lung respiratory compartments and AdC cells demonstrated in this study will be informative for the molecular diagnosis of lung AdC.

Amplification and expression of a cellular oncogene (c-myc) in human gastric adenocarcinoma cells.

Three of 16 human gastric adenocarcinoma samples, maintained as solid tumors in nude mice, were found to carry amplified c-myc genes. In two samples with a high degree of c-myc DNA amplification (15- to 30-fold), double minute chromosomes were observed in karyotype analysis. The level of c-myc RNA was markedly elevated in a rapidly growing and poorly differentiated tumor, whereas it was only slightly elevated in a slowly growing and more differentiated tumor.

Sp1-mediated transcription of the Werner helicase gene is modulated by Rb and p53.

The regulation of Werner's syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5' upstream region (2.8 kb) of WRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides -67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRN promoter-luciferase reporter (WRN-Luc) plasmids that contained the 5'-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2. 5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability.

Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA.

Adenine paired with 8-hydroxyguanine (oh(8)G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells. Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His(6)-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh(8)G and A:G mismatches by DNA cleavage assay and gel mobility shift assay. We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q(324) and 2-Q(310) proteins with type 1-H(324) and 2-H(310) proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain. In a reaction buffer with a low salt (0-50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh(8)G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh(8)G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh(8)G, was proportionally reduced. The glycosylase activity on A:oh(8)G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins. These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh(8)G under physiological salt concentrations.

Alterations of glycolipids of human leukemia cell line HL-60 during differentiation.

Alterations of glycolipids of human leukemia cell line HL-60 during differentiation were investigated. The neutral glycolipids of HL-60 cells are of the lacto-types such as lactosylceramide (LacCer) and lactoneotetraglycosylceramide (nLcOse4Cer). The gangliosides of HL-60 cells consisted of a mixture of the lacto-types and the ganglio-types. Chemical inducers of differentiation, 12-O-tetradecanoylphorbol 13-acetate (TPA) and dimethyl sulfoxide, altered the glycolipid profile; II3-alpha-acetylneuraminyl-lactosylceramide (GM3) increased in amount and LacCer and nLcOse4Cer decreased. Tunicamycin had no effect on the glycolipid synthesis. The GM3 accumulated more than tenfold in the TPA-induced macrophage-like cells. This accumulation could be due to the increased activity of LacCer sialytransferase.

Loss of heterozygosity on chromosomes 1p and 11p in sporadic pheochromocytoma.

We used 29 polymorphic DNA markers to analyze tumor DNA samples from six patients with sporadic pheochromocytoma for possible loss of chromosomal heterozygosity; four had benign disease and two had malignant disease. Loss of heterozygosity was observed on four chromosomes: 1p (three of four patients), 2p (one of one), 5q (two of six), and 11p (three of five). Chromosomes 1p and 11p frequently had allelic deletions in these tumors, and these deletions may play an important role in the development of pheochromocytoma.

Detection of novel germ-line p53 mutations in diverse-cancer-prone families identified by selecting patients with childhood adrenocortical carcinoma.

BACKGROUND: Germ-line p53 mutations appear to be inherited among the members of families diagnosed with Li-Fraumeni syndrome (LFS). The mutations detected in those families to date have been clustered in exon 7 of the p53 gene and, typically, have been single-base substitutions resulting in amino acid changes. PURPOSE: Our aim was to define the spectrum of p53 mutations associated with LFS. METHODS: From seven cancer-prone families identified by selecting members with childhood adrenocortical carcinoma as probands, we chose two families, each of which had two members from whom specimens could be obtained for genetic analysis. To detect germ-line p53 gene mutations in these individuals, we performed polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis with Taq polymerase and oligonucleotide primers specific for p53 gene sequences. Genomic DNA extracted from fresh tissue samples and paraffin-embedded tumor samples was amplified, denatured, and electrophoresed on neutral polyacrylamide gels. PCR amplification was also carried out using total RNA from adrenocortical carcinoma samples of the proband in family 1. PCR products were purified, subcloned, and sequenced. RESULTS: We detected novel germ-line p53 mutations in affected members of both cancer-prone families. In the proband of family 1, a single-base deletion was detected at the first nucleotide of codon 307 in exon 8 of the p53 gene, resulting in a premature stop codon in exon 10. In family 2, we detected an A to C transversion at the second nucleotide of codon 286 in exon 8, both in DNA isolated from the adrenocortical tumor of the proband and in DNA isolated from the astrocytoma of the proband's father. This single-base substitution resulted in an amino acid substitution of alanine for glutamic acid. Both of these mutations are located outside the highly conserved region of the p53 gene where mutations in patients with LFS have been reported previously. CONCLUSION: Our results indicate that a wide range of germ-line p53 mutations is inherited in members of diverse-cancer-prone families.

Frequent allelic losses on chromosomes 2q, 18q, and 22q in advanced non-small cell lung carcinoma.

Although it is widely accepted that tumor suppressor genes play an important role in the genesis and progression of human cancer, little is known about genetic events that accumulate during multistage lung carcinogenesis. Thus, to determine a subset of tumor suppressor genes that are involved in the genesis and progression of non-small cell lung carcinoma (NSCLC), 22 brain metastases and 23 stage I primary lung tumors were examined for allelic losses at 40 loci on 10 chromosomes including the loci of 5 tumor suppressor genes, APC, WT1, RB, p53, and DCC. The incidence of allelic losses on chromosomes 3p, 13q, and 17p was high (> 60%) in both primary tumors and brain metastases. In brain metastases, a high incidence of allelic losses (> 60%) was also observed at loci on chromosomes 2q, 18q, and 22q, and the incidence of allelic losses on these chromosomes in brain metastases was significantly higher than that in primary tumors (P < 0.05). In two cases of brain metastases with corresponding primary lung tumors, sequential accumulation of allelic losses during progression of primary lung tumors was observed on several chromosomes including chromosomes 2q and 18q. These results indicate that, besides loss of heterozygosity for chromosomes 3p, 13q, and 17p, loss of heterozygosity for chromosomes 2q, 18q, and 22q also occurs frequently in advanced NSCLCS. Thus, it is possible that loss of heterozygosity on chromosomes 2q, 18q, and 22q occurs late in the progression of NSCLC and/or causes phenotypic alterations of NSCLC cells into more aggressive ones.

Infrequent loss of chromosomal heterozygosity in human stomach cancer.

By molecular genetic approach using polymorphic DNA markers which detect allelic deletion at specific chromosomal loci, we analyzed 30 human stomach cancers for possible loss of chromosomal heterozygosity. We analyzed 25 loci on 18 different chromosomes covering regions frequently deleted in several types of cancers. Loss of chromosomal heterozygosity was observed only in five of 30 cases examined, and it was infrequently detected at 10 loci on seven different chromosomes including chromosome 1 in two of 12 cases, chromosome 12 in one of four cases and chromosome 13 in three of 27 cases. It was also observed at loci on chromosomes 11, 14, 16, and 19 with very low frequency (less than 10%), but not on other chromosomes: chromosomes 3, 5, 6, 9, 10, 15, 17, 18, 20, and 22. Thus, in human stomach cancer, loss of heterozygosity occurs infrequently even at chromosomal loci often deleted in other types of cancers.

Alterations of the PPP1R3 gene in human cancer.

Recently, the PTEN/MMAC1 gene encoding a protein phosphatase (PP) and the PPP2R1B gene encoding a regulatory subunit of PP2A have been identified as being genetically altered in several types of human cancers, indicating that aberrations of intracellular signaling pathways via PPs are involved in human carcinogenesis. Here we report genetic alterations of the PPP1R3 gene located at chromosome 7q31, which encodes regulatory subunit 3 of PP1, in various types of human cancers. Mutations of the PPP1R3 gene were detected in 5 of 33 (15%) non-small cell lung cancer cell lines and 2 of 38 (5%) primary non-small cell lung cancers and were also observed in cell lines derived from a small cell lung cancer, an ovarian cancer, a colorectal cancer, and a gastric cancer. Mutations were widely dispersed in the coding region of the PPP1R3 gene. Three of the 11 detected mutations were nonsense mutations, whereas the remaining ones were missense mutations, most of which caused substitutions of evolutionarily conserved amino acids. These findings suggest that PPP1R3 alteration plays a role in the development of human cancers and that PPP1R3 could act as a tumor suppressor gene.

Association of multiple copies of the c-erbB-2 oncogene with spread of breast cancer.

Amplification of c-erbB-2 was at least three times more frequent in breast cancer than in most other types of carcinoma, and was not found in sarcomas or hematological malignancies. Amplification of c-erbB-2 was found in 15 of 86 primary breast cancers and in 3 of 12 secondary breast cancers. Amplification was more common in breast tumors of advanced stage, and in tumors which had metastasized to regional lymph nodes sites. Gene amplification was observed in 21% (4 of 19) of primary tumors which recurred within 3 years of mastectomy and in 6% (2 of 32) of nonrecurrent tumors.

Clinicopathological significance of Fhit protein expression in stage I non-small cell lung carcinoma.

Abnormalities in structure and expression of the FHIT gene have been detected in a considerable fraction of primary lung tumors. Previous reports indicated that FHIT gene alterations can be simply detected by immunohistochemical methods. Therefore, we investigated the association of Fhit expression with clinicopathological features and allelic imbalance (AI) at the FHIT locus in 105 stage I non-small cell lung cancers (NSCLC) by the immunohistological method and PCR analysis. Thirty-six of 105 (34%) tumors showed marked reduction of Fhit immunoreactivity. Fhit expression was markedly reduced in most squamous cell carcinomas (24 of 28, 86%), whereas such a reduction was detected only in a small subset of adenocarcinomas (7 of 67, 10%; P < 0.001). A marked reduction of Fhit protein expression was observed more frequently in patients with a smoking history (32 of 80, 40%) than in patients without a smoking history (4 of 25, 16%; P = 0.013). These results indicate that FHIT gene alterations preferentially occur in squamous cell carcinomas and in smokers. Furthermore, a reduction of Fhit protein expression in tumor cells was associated with a poorer survival of patients with stage I NSCLC, irrespective of histological subtypes of tumors (P = 0.005; log-rank test). Fhit expression was reduced preferentially in tumors with AI at the FHIT locus; however, AI at the FHIT locus did not correlate with patients' survival (P = 0.262; log-rank test). These results suggested that Fhit protein expression could be a useful molecular marker for the prognosis of patients with surgically resected stage I NSCLC.

Deletion map of chromosome 9 and p16 (CDKN2A) gene alterations in neuroblastoma.

We reported previously that loss of heterozygosity (LOH) on chromosomes 2q, 9p and 18q frequently occurs in neuroblastoma and that patients with 9p LOH in the tumors showed statistically significant association with an advanced stage of the disease and poor prognosis. To determine the role of chromosome 9 loss in neuroblastoma, we performed deletion mapping of chromosome 9 in 80 cases of neuroblastoma using 11 polymorphic microsatellite markers and a restriction fragment length porymorphism marker. LOH at one or more loci on chromosome 9 was detected in 33 of 80 cases (41%). Chromosome 9p was lost in 24 of 80 cases (32%), whereas chromosome 9q was lost in 18 of 80 cases (23%). There were two commonly deleted regions mapped to 9p21 between the D9S171 marker and the IFNB1 marker and 9q34-qter distal to the D9S176 marker. In addition, patients with LOH at 9p21 but not at 9q34-qter in the tumors showed statistically significant association with poor prognosis (P = 0.023). Because the commonly deleted regions at 9p21 includes the p16 (CDKN2A) gene, the status of the p16 gene was further examined in 80 fresh tumors and 19 cell lines of neuroblastoma. A missense mutation was detected at codon 52 in a fresh tumor. The p16 gene was not expressed in 13 of 19 cell lines (72%), and 5 of the 13 cell lines displayed methylation of the CpG island surrounding the first exon of the p16 gene. These results suggest that the p16 gene is a candidate tumor suppressor gene for neuroblastoma, and its inactivation may contribute to the progression of neuroblastoma.

Analysis of replication error (RER+) phenotypes in cervical carcinoma.

Infection of epithelial cells with human papillomavirus is an important early event in the development of cervical dysplasia. However, progression to overt malignancy appears dependent upon further genetic and/or epigenetic events. We have recently developed methodologies for the simultaneous analysis of loss of heterozygosity (LOH) at multiple PCR-based microsatellite loci using semiautomated fluorescent DNA sequencing technology to determine the locations of tumor suppressor genes which are inactivated during tumor progression. While examining 30 microsatellite loci for LOH on chromosomes 3p, 4, and 11q, we detected novel tumor-specific alleles indicative of microsatellite instability (MI). The methodology allowed rapid and accurate comparison of over 3000 genotypes from 89 primary tumors and 10 cervical carcinoma-derived cell lines and showed that five tumors (5.6%) and one human papillomavirus-negative cell line, C33A, had genetic features consistent with a replication error (RER+) phenotype as defined by MI at two or more loci. In each of the RER+ tumors, LOH was also observed at one or more loci on each of the three chromosomes examined. These findings suggest that defects in DNA repair-associated genes are rarely acquired and do not supersede allelic loss during cervical carcinogenesis. In addition, the semiautomated multiplex approach has proven unequivocal in the detection and interpretation of MI and should greatly accelerate the rapidity and accuracy of analysis of such defects in tumors. Moreover, the number of loci that can be relatively easily examined in this way will also allow a detailed statistical consideration of the importance of such events.

Increased formation of oxidative DNA damage, 8-hydroxydeoxyguanosine, in human livers with chronic hepatitis.

8-Hydroxydeoxyguanosine (oh8dG) is a promutagenic DNA lesion produced by oxygen radicals. We examined alterations in the oh8dG level in human livers which have chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The oh8dG content in livers with chronic hepatitis was significantly higher than the oh8dG content in normal livers (P < 0.05). There was also a significant correlation between the oh8dG content in noncancerous liver tissues with individual serum alanine aminotransferase concentration (r = 0.515; P < 0.001). Thus, chronic inflammation in the liver produces oxidative DNA damage, which may increase the risk for genomic alterations causing hepatocarcinogenesis.

Frequent loss of heterozygosity on chromosome 14q in neuroblastoma.

Using 29 polymorphic DNA markers which detect allelic deletion of genes at specific loci on 19 different chromosomes, we analyzed 14 neuroblastomas for possible loss of chromosomal heterozygosity. The incidence of loss of heterozygosity was high at the D14S1 locus on chromosome 14q, being detected in six of 12 patients (50%). In spite of the cytogenetic finding suggesting high frequency of chromosome 1p deletion, loss of heterozygosity at the MYCL locus on 1p32 was detected only in two of nine patients (22%). It was also found in two of 11 patients (18%) on 13q, but not on chromosomes 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, and 20. The present results indicate that recessive genetic changes involving sequences on chromosome 14q may play an important role in the development of neuroblastoma.

Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus.

To understand the involvement of allelic losses and inactivation of tumor suppressor genes for the development of endometrial carcinoma of the uterus (EC), 24 cases of EC were examined for loss of heterozygosity (LOH) using a total of 57 polymorphic DNA markers covering all 23 pairs of chromosomes. LOH was observed at 27 loci on 10 different chromosomes, i.e., chromosomes 1, 3, 6, 11, 13, 15, 17, 18, 20, and 21, but was not detected at loci on chromosomes 4, 5, 7, 9, 10, 12, 14, 16, and X. It was observed only in seven of 24 cases, and the other 19 cases did not show LOH at any loci examined, including five cases of tumors with a high proportion of adenomatous hyperplasia. Among seven tumors with LOH at one or more loci, five tumors showed LOH at loci on the short arm of chromosome 17. Furthermore, mutations of the p53 gene, which is located on the short arm of chromosome 17, were detected in three of these 24 tumors by a polymerase chain reaction-single strand conformation polymorphism analysis and subsequent DNA sequencing. In two of these three tumors, p53 mutations were accompanied by the loss of wild-type p53 alleles. These results suggest that inactivation of the p53 gene is involved in the development of EC as in the case of several other types of human cancers.