PTCH gene mutations in invasive transitional cell carcinoma of the bladder.

LOH analysis suggests that multiple tumor suppressor genes play a role in the development of human TCC. The human homolog of the Drosophila PTCH was recently cloned and mapped to the BCNS locus on 9q22.3, a chromosomal region commonly deleted in TCCs. We first examined the steady state mRNA transcription of the PTCH, SMOH and GLI3 genes of the HH signal transduction pathway in TCC cell lines and normal urothelium. Normal urothelium and TCC cell lines express these three genes within the PTCH signal transduction pathway. We then screened for PTCH mutations in 'hot spot' exons 6, 8, 13 and 16 by PCR/SSCP analysis of genomic DNAs from 54 TCC tumor samples and control autologous peripheral blood lymphocytes. DNA sequence analysis confirmed TCC-specific mutations in two of 54 patients (3.7%). These mutations resulted a single amino acid substitution and two frame shifts. One tumor had PTCH mutations in exon 16 as well as exon 13 and one tumor had a mutation in exon 13 alone. Both TCC tumors that contained PTCH mutations had a loss of heterozygosity at 9q. Although the PTCH protein has an unknown function in urothelial cells, the detection of the PTCH, SMOH and GLI3 transcripts in normal urothelium and TCC cell lines and rare PTCH mutations in tumor samples suggest that the HH pathway may have a role in controlling the proliferation of urothelial cells and that PTCH mutations may contribute to the development of a subset of TCCs.

Melanoma cell lines from different stages of progression and their biological and molecular analyses.

The biological and molecular characteristics of cell lines from metastatic melanomas have been extensively studied but less is known about cells from the biologically earliest stage of primary melanoma. The overall success rate of establishing permanent cell lines from such lesions is only 10% of that for biologically late primary or metastatic melanomas, although our laboratory now has eight cell lines available. The cells are immortal but show reduced or no proliferation in soft agar and immunodeficient mice when compared with primary melanomas from the biologically advanced vertical growth phase. Metastatic melanoma cell lines from patients with familial melanoma or xeroderma pigmentosum are biologically similar to those from patients with spontaneous melanomas. Irrespective of the malignant stages, deletions and mutations can occur in exons 1-3 of the p16INK4A gene. DNA fingerprinting was then employed to demonstrate the uniqueness of individual cell lines and to confirm the identity of cell lines derived from same patients. These cell lines are an excellent resource to investigate melanoma progression.

Novel p21WAF1/CIP1 mutations in superficial and invasive transitional cell carcinomas.

Alterations in the p53 gene are a predominant component in the development of transitional cell carcinoma (TCC), but the particular pathways distal to p53 alterations which contribute to urothelial transformation are not defined. Here, the p21WAF1/CIP1 gene, a p53 inducible and p53 independent gene product, was studied in TCC. p21WAF1/CIP1 expression was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) from five cell lines and 28 tumor specimens (14 superficial, 14 muscle invasive). This was expressed as a ratio of the gene product to L7, a ribosomal housekeeping gene. In addition, exons 4 through 8 of the p53 gene as well as exon 2 of the p21WAF1/CIP1 gene were assayed for mutations by polymerase chain reaction/single stranded conformation polymorphism analysis (PCR/SSCP). Candidate mutations were verified by sequencing. p21WAF1/CIP1/L7 expression was significantly decreased in invasive lesions compared to superficial lesions (P<0.002). p53 mutations were detected by PCR/SSCP in seven tumors [25%] (one superficial, six invasive) and p21WAF1/CIP1/L7 expression was significantly decreased in all tumors that had p53 mutations (P<0.007). PCR/SSCP analysis of exon 2 in p21WAF1/CIP1 detected band shifts in four/28 tumor specimens (two superficial, two invasive), which sequencing and comparison to autologous normal matched DNA revealed as novel mutations.

Three new dinucleotide repeat polymorphisms on human chromosome 9: D9S970, D9S971, and D9S972.

Three human chromosome 9-specific cosmid recombinants containing (CA)n microsatellites are described. Three microsatellite loci, D9S970, D9S971, and D9S972, were observed to have heterozygosities of 0.78, 0.84, and 0.82, respectively. Subchromosomal localizations were determined by R-banding and fluorescence in situ hybridization.

Chromosome 9 deletion in sporadic and familial melanomas in vivo.

Twenty microsatellite loci on chromosome 9 were analysed for allelic losses in DNAs from 30 uncultured melanomas from 25 patients, relative to DNA from autologous peripheral blood lymphocytes. All patients were constitutionally heterozygous at several loci, and loss of heterozygosity (LOH) affecting 9p was observed in melanoma DNAs from 18 individuals (72%). Observations of losses of identical alleles in different metastatic lesions from the same patients, and of LOH in a vertical growth phase primary melanoma, were consistent with previous reports of chromosome 9 deletion early in melanoma development. LOH data suggested the loss of entire copies of chromosome 9 in 11 cases, and the terminal deletion of all or a portion of 9p in six cases. A somatic interstitial deletion of 9p between D9S162 and D9S169 was seen in a familial melanoma. This 21 cM deleted region corresponded with the previously reported positions of homozygous deletions in melanoma cell lines, and of the familial melanoma susceptibility locus (MLM). As 16 of the 18 cases of 9p LOH in the present study were observed in individuals with no family history of melanoma, it is likely that the MLM locus plays a role in the development of most sporadic melanomas.

Chromosome 10 allelic loss in malignant melanoma.

The involvement of tumor suppressor genes in the progression of melanoma has been suggested by the frequent deletion of specific regions of the genome in melanoma. In this study, a panel of 18 surgically removed melanomas from 15 patients was analyzed for loss of heterozygosity (LOH) at 10 polymorphic loci on chromosome 10. LOH was observed in 7 (50%) of 14 informative patients. LOH data suggested that melanomas from 5 patients had lost entire copies of chromosome 10, and that melanomas from 2 patients had lost copies of 10q. In contrast, LOH was not observed on chromosome 15, 20, or 21. These results are consistent with previous cytogenetic observations and provide indirect evidence that there is a tumor suppressor gene on the long arm of chromosome 10 which is relevant to melanoma development.

Characterization of chromosome 9 deletions in transitional cell carcinoma by microsatellite assay.

A panel of 18 superficial or invasive transitional cell carcinomas (TCCs) was analyzed for chromosome 9 deletions by performing a high-density loss of heterozygosity (LOH) analysis. Twenty-five microsatellite loci were assayed by the polymerase chain reaction (PCR) and 7 restriction fragment length polymorphism (RFLP) loci were analyzed by Southern blotting. Concordant results were obtained with these methods, including direct comparisons at 2 loci. Chromosome 9 LOH was observed in 13 (72%) of 18 informative cases, including 10 superficial lesions. In contrast, LOH on chromosomes 10, 15, 20 and 21 was < or = 8%. Evidence for missing copies of chromosome 9 was observed in 7 of 13 LOH cases. Comparison of cases with subchromosomal LOH implicated the region between the D9S55 locus on 9p12 and the argininosuccinate synthetase (ASS) locus on 9q34.1 as the location of a tumor suppressor gene relevant to TCC.

Retroposition in a family of carcinoma-associated antigen genes.

The gene encoding the carcinoma-associated antigen defined by the monoclonal antibody GA733 is a member of a family of at least two type I membrane proteins. This study describes the mechanism of evolution of the GA733-1 and GA733-2 genes. A full-length cDNA clone for GA733-1 was obtained by screening a human placental library with a genomic DNA probe. Comparative analysis of the cDNA sequence with the previously determined genomic sequence confirmed that GA733-1 is an intronless gene. The GA733-2 gene encoding the monoclonal antibody-defined antigen was molecularly cloned with a cDNA probe and partially sequenced. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons. The putative promoter regions of the GA733-1 and GA733-2 genes are unrelated. These findings suggest that the GA733-1 gene was formed by the retroposition of the GA733-2 gene via an mRNA intermediate. Prior to retroposition, the GA733-2 gene had been affected by exon shuffling. Analysis of GA733-2 exons revealed that many delineate structural motifs. The GA733-1 retroposon was localized either to chromosome region 1p32-1p31 or to 1p13-1q12, and the GA733-2 founder gene was localized to chromosome 4q.

Molecular cloning of murine monoclonal anti-idiotypic Fab.

Anti-idiotypic antibodies (Ab2) binding to idiotopes on antibodies with various antigen binding specificities (Ab1) are potential regulators of immunity in a variety of diseases, such as autoimmunity, cancer, and viral, bacterial, or parasitic infections. Furthermore, Ab2 are useful probes for the characterization of receptor/ligand interactions. Thus far, Ab2 production has been limited to the isolation of polyclonal Ab2 from immune sera or monoclonal Ab2 from hybridoma supernatants. However, both approaches have produced a limited number of Ab2. As an alternative approach, we demonstrate here the production of Ab2-Fab by using repertoire cloning. Using HIV-1 as a model system, the Ab2-Fab were generated from the spleens of mice immunized with the virus-neutralizing and syncytia-inhibiting anti-HIV-1 monoclonal antibody 0.5 beta. A bacteriophage lambda vector system was used to express a combinatorial library in Escherichia coli. Iodinated 0.5 beta was used to identify 17 Ab2-Fab clones. DNA sequence analysis of five clones revealed three similar kappa and Fd combinations. The Ab2-Fab bound with high affinity (3.5-6.5 x 10(9) liters/mol) specifically to the Ab1 and not to isotype-matched antibodies with unrelated specificities. The three Ab2-Fab probably bind to the same idiotope on the Ab1 as demonstrated in cross-competition binding studies. The Ab2-Fab inhibited binding of the Ab1 to antigen, and therefore, may functionally mimic the epitope defined by the Ab1. Repertoire cloning of Ab2-Fab may facilitate the generation of Ab2 that have potential as modulators of immune responses against various antigens.

Baculovirus recombinant expressing a secreted form of a transmembrane carcinoma-associated antigen.

GA733-2 is a monoclonal antibody-defined, 40-kDa glycoprotein antigen that is associated with carcinomas of various origins. Hydrophobicity analysis of the protein sequence predicted by complementary DNA (cDNA) has suggested that the GA733-2 antigen is a type I membrane protein. In this study, the polymerase chain reaction was used in a strategy to omit cDNA sequences for the transmembrane and cytoplasmic domains, thereby converting the extracellular domain into a secretory protein. Full-length and truncated cDNAs were cloned into the baculovirus transfer vector pVL1392 and introduced into Autographa californica nuclear polyhedrosis virus by homologous recombination. The full-length cDNA baculovirus recombinant directed the expression of a 40-kDa glycoprotein that was confined to infected Spodoptera frugiperda cells, whereas cells infected with the truncated cDNA baculovirus recombinant abundantly secreted a 31-kDa glycoprotein into the culture medium. Recombinant secretory antigen displayed an in vitro immunoreactivity to monoclonal antibody and an in vivo immunogenicity in mice that were similar to native antigen. The facile purification of mg quantities of carcinoma-associated antigen will enable an evaluation of its immunogenicity in cancer patients.

Growth-factor-independence and invasive properties of colorectal carcinoma cells.

During serial passage of the colorectal carcinoma cell line SW1116 in athymic nude mice, we selected 2 variants that metastasized to the lungs and liver. The metastatic capacity of these in vivo variant cell lines was associated with their ability to (1) grow under growth-factor-deprived conditions, (2) invade and transgress a reconstructed basement membrane with high effectiveness, and (3) produce higher activities of the substrate-degrading enzymes collagenase and plasminogen activator as compared to parental cells. To assess the relative contribution of growth-factor-independence and high levels of invasiveness/motility to the metastatic phenotype, variants of 6 colorectal carcinomas were selected in vitro by adaptation to a growth-factor-free culture medium followed by selection of highly invasive cells in chemoinvasion assays. Four out of 6 cell lines selected for growth-factor-independence showed significantly higher levels of invasiveness through reconstructed membranes, suggesting co-segregation of growth-factor-independence and high levels of invasiveness in vitro. Using an in vitro chemoinvasion assay, 2 poorly and 1 highly invasive cell line were further selected for invasiveness. After 6 selection passages, all cell lines were highly invasive and showed high motility rates. However, when injected s.c. into athymic nude mice to test their metastatic capacity in vivo, double-selected variant cell lines did not form spontaneous metastases. Our results indicate that growth-factor-independence and high levels of invasiveness, although associated with the metastatic phenotype, are not sufficient for experimental metastasis formation of colorectal carcinoma cells in vivo.

The exon-intron organization of the human erythrocyte alpha-spectrin gene.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens.

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

Molecular cloning of cDNA for the carcinoma-associated antigen GA733-2.

Defined by monoclonal antibody GA733, the GA733-2 antigen is a cell surface 40-kDa glycoprotein associated with human carcinomas of various origins. Molecular clones for the GA733-2 antigen were isolated from a colorectal carcinoma cell line cDNA library using the high-efficiency COS cell expression system. A 1.4-kilobase cDNA species was enriched by immunoselection with monoclonal antibody. The authenticity of individual clones was established by immunologic and sequence criteria. At the amino acid sequence level, GA733-2 was found to be greater than 99% identical to the previously described KSA antigen defined by monoclonal antibody KS1/4. The amino acid sequence derived from the previously described GA733-related gene, GA733-1, was found to be 49% identical to GA733-2. The positions of 12 cysteine residues in the extracellular domains of the two GA733 antigens are conserved, as is the overall distribution of hydrophobic and hydrophilic residues. A 1.45-kilobase transcript of the GA733-2/KSA gene was found to be expressed in cell lines derived from colorectal and pancreatic carcinoma.

The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.

Molecular cloning of the breakpoint region on chromosome 6 in cutaneous malignant melanoma: evidence for deletion in the c-myb locus and translocation of a segment of chromosome 12.

Nonrandom involvement of chromosome 6 in cutaneous malignant melanoma have been noted by several investigators. Recently an alteration in the c-myb locus (6q22-23) has been identified by Southern analysis in the WM983A cell line which was derived from a primary melanoma of the vertical growth phase. In the present study, the nature of this rearrangement in the WM983A cell line has been further characterized by molecular cloning and nucleotide sequence analysis of the break-point region in the c-myb locus. The results of this investigation demonstrate that the rearrangement in the 6q22-23 region results in deletion of the 3'-end of the c-myb locus with the concomitant translocation of a portion of chromosome 12 to chromosome 6.

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.

Tumor progression in the human melanocytic system.

The isolation and routine tissue culture of melanocytic cells from normal skin, precursor nevi, primary and metastatic melanomas has allowed the experimental study of different stages of tumor progression. Characteristic differences between cultured normal melanocytes and highly malignant metastatic melanoma cells were: 1) limited life span for normal melanocytes and non-malignant nevus cells versus infinite growth for malignant melanoma cells; 2) inability to grow anchorage-independently versus high colony forming-efficiency in soft agar; 3) non-tumorigenicity versus tumorigenicity in athymic nude mice; 4) dependence on exogenous growth factors and other mitogens versus autonomous growth in protein-free medium; 5) expression of melanocyte-associated antigens versus expression of melanoma-associated antigens; and 6) diploid karyotype versus non-random chromosomal abnormalities. The only major distinction found between advanced primary and metastatic melanomas was that only metastatic melanoma cells proliferated continuously in the absence of growth factors or other proteins. However, advanced primary melanoma cells could be clearly distinguished from dysplastic nevus cells by their growth behavior and growth factor requirements. Only limited information is available on the biologic, genetic, immunologic and molecular properties of dysplastic nevus cells and early (radial growth phase) primary melanoma cells but these cells appear to differ markedly from advanced primary and metastatic cells. The availability of cells from sequential steps of tumor progression in the human melanocytic system offers a unique experimental model for the study of malignant transformation.

Sequence investigation of the major gastrointestinal tumor-associated antigen gene family, GA733.

The monoclonal antibody-defined, tumor-associated antigen GA733 was purified from the SW948 human colorectal carcinoma cell line and its partial amino acid sequence was determined. By using a synthetic oligonucleotide probe, two recombinants were isolated from a total human genomic library. We prove the existence of a family of GA733 genes. One of the genomic isolates is demonstrated to be an intronless gene, which is transcribed in pancreatic carcinoma cell lines and in placenta. The GA733 proteins were observed to contain sequences homologous to a repeat unit occurring 10 times in thyroglobulin and once in the HLA-DR-associated invariant chain. A more evolutionarily distant relationship was found with the alpha chain of the interleukin 2 growth factor receptor.