Expression of an isoform of the novel signal transduction protein ST5 is linked to cell morphology.

The human ST5 gene is expressed as 4.6, 3.1 and 2.8 kb transcripts encoding putative 126, 82 and 70 kDa proteins that function in the MAP kinase signaling pathway in transient expression assays. Expression of the 2.8 kb transcript correlates with reduced tumorigenicity in HeLa-fibroblast hybrids, suggesting a role in tumor suppression. We now report the detection of ST5 proteins in cellular extracts, demonstrate specific expression of p70 in non-tumorigenic HeLa-fibroblast hybrids, extend the correlation between p70 expression and cellular morphology to a wide variety of cell lines, and provide direct evidence that p70 can effect changes in cell growth and morphology. ST5 proteins were identified in extracts of human, mouse and simian epithelial cells and fibroblasts, but were absent from lymphoid cells. Transfection of the 2.8 kb cDNA into a p70-negative mouse fibroblast line yielded stable transfectants with a flattened, less refractile morphology relative to controls. The p70 expressing clones had initial growth rates similar to those of control cells but their saturation density was reduced threefold, suggesting a restoration of contact-regulated growth. In conjunction with previous findings, these results suggest that ST5 proteins participate directly in events affecting cytoskeletal organization and tumorigenicity.

Squamous cell papillomas of the esophagus: a study of 23 lesions for human papillomavirus by in situ hybridization and the polymerase chain reaction.

This study assessed squamous cell papillomas of the human esophagus for the presence of human papillomavirus (HPV) and correlated the results with histological features. Twenty-three lesions obtained by endoscopic biopsy from 17 patients were studied, first by in situ hybridization (ISH) for HPV types 6-11, 16-18, 18, and 31-33-51, and second by the polymerase chain reaction (PCR) with amplification of multiple HPV types and demonstration of amplified product by ethidium bromide staining and Southern blot hybridization for HPV types 6-11, 16, and 18 in each case. Evidence of HPV DNA was found in only one lesion, which showed HPV type 6-11 by ISH and HPV positivity by Southern blotting of the amplified product after the PCR. This case exhibited histological features suggestive of HPV infection, although no morphological changes specific to the lesion were identified. The remaining 22 lesions, including those from cases in which multiple papillomas were present, were negative for HPV. The results show that HPV DNA is frequently not detectable in esophageal squamous cell papillomas, even when highly sensitive techniques are used. These findings are consistent with the hypothesis that other pathogenetic mechanisms, such as mucosal injury and repair, are important in the etiology of these lesions.

Enhanced sensitivity with a novel TCRgamma PCR assay for clonality studies in 569 formalin-fixed, paraffin-embedded (FFPE) cases.

BACKGROUND: Clonal rearrangement of genes encoding the immunoglobulins (Ig) and T-cell antigen receptors (TCR) are considered to be useful markers for the diagnosis of lymphoma and for determining the clonal origins of B- and T-cell populations in lymphoid neoplasms. METHODS AND RESULTS: Polymerase chain reaction-based clonality assays for TCRgamma, TCRbeta, and immunoglobulin (Ig) heavy chain (IgH) gene rearrangements were evaluated for diagnostic sensitivity and specificity in 569 formalin-fixed, paraffin-embedded (FFPE) tissues. Combined TCRbeta and TCRgamma assays enhanced the routine detection of TCR clonality to 90% of all peripheral T-cell lymphoma (PTCL) cases. IgH clonality was detected in 59% of 241 peripheral B-cell lymphoma (BCL) cases and 6% of 169 PTCL cases. Of 452 lymphomas, 5% could not be classified phenotypically as B or T lineage after immunohistochemical and clonality studies. Of all BCL cases analyzed, 24% had detectable TCRbeta and/or TCRgamma clonality. Of these BCL with biclonal results, 47% were extranodal lymphomas from skin and various tissues. CONCLUSIONS: Clonality assays were useful for distinguishing reactive or benign lymph nodes from neoplastic lymphoid infiltrates in most cases. The inclusion of TCRb and TCRg assays in the assessment of lymphomas results in a significant increase in the sensitivity of clonality detection, but is of limited utility in assessing the T- or B-cell phenotype of the tumor.

Morbilliviral epizootic in bottlenose dolphins of the Gulf of Mexico.

Morbillivirus infection was diagnosed in 35/67 bottlenose dolphins (Tursiops truncatus) from the Gulf of Mexico that stranded from October 1993 through April 1994 in Alabama, Mississippi, and Texas (USA) during periods of increased dolphin strandings in each of the 3 states. Diagnosis was based on histologic lesions, immunohistochemical demonstration of mobilliviral antigen, and detection of morbilliviral RNA by a reverse transcriptase polymerase chain reaction (RT-PCR) test performed on formalin-fixed, paraffin-embedded tissue (5 dolphins), on histologic lesions and detection of morbilliviral RNA by RT-PCR performed on formalin-fixed, paraffin-embedded tissue (1 dolphin), and on detection of morbilliviral RNA by RT-PCR performed on unfixed lung samples collected from carcasses with advanced postmortem autolysis (29 dolphins). Histologic lesions included proliferative interstitial pneumonia with syncytial cells and eosinophilic intranuclear and intracytoplasmic inclusion bodies, lymphoid depletion and syncytial cells with eosinophilic intranuclear inclusion bodies in lymph nodes, eosinophilic intracytoplasmic inclusion bodies in transitional epithelium of urinary bladder, and a syncytial cell with eosinophilic intranuclear inclusion bodies in epidermis. Concomitant pulmonary aspergillosis was diagnosed histologically in 4 dolphins. This is the 5th reported morbilliviral epizootic of aquatic mammals and the 2nd involving bottlenose dolphins in the United States.

Postmortem diagnosis of morbillivirus infection in bottlenose dolphins (Tursiops truncatus) in the Atlantic and Gulf of Mexico epizootics by polymerase chain reaction-based assay.

Lung tissue from 39 bottlenose dolphins (Tursiops truncatus) found dead off the U.S. Atlantic and Gulf of Mexico coasts from 1987 to 1994 was examined for the presence of morbillivirus using a reverse transcriptase polymerase chain reaction (RT-PCR) technique. Of the Atlantic cases examined, six of six were positive using this assay; 18 of 25 Gulf of Mexico cases with amplifiable RNA also were found to be positive, and eight additional specimens had no amplifiable RNA. The RT-PCR allowed the diagnosis of morbillivirus infection to be made from either sections of paraffin-embedded formalin-fixed material or from unfixed tissue. Conformation of diagnosis was made by subsequent hybridization of the amplified products with a dolphin morbillivirus specific probe using the Southern blot technique. Application of this method to autolyzed post-mortem tissues allows diagnoses of morbillivirus infection to be made in specimens which cannot be evaluated by histologic and immunocytochemical techniques.

Deletion of the COOH terminus converts the ST5 p70 protein from an inhibitor of RAS signaling to an activator with transforming activity in NIH-3T3 cells.

Expression of the human protein ST5-p70 correlates with reduced tumorigenic phenotype in mammalian cells, reverts their transformed phenotype, and restores their contact-dependent growth. Furthermore, expression of p70 in COS-7 cells suppresses activation of mitogen activated protein kinase MAPK/ERK2 by the largest ST5 product, p126, in response to epidermal growth factor stimulation. Here we show that deletions of the COOH-terminal region of p70 transform NIH3T3 cells and induce their anchorage-independent growth. Analysis of signaling leading to MAPK/ERK2 stimulation revealed that in COS-7 cells, expression of either p70-DeltaC1 or p70-DeltaC2 markedly enhanced ERK2 activity in a growth factor-independent manner. Whereas wild-type p70 slightly inhibited ERK2 activation by RAS and MEK2, co-expression or p70-DeltaC1 or p70-DeltaC2 with either protein stimulated ERK2 cooperatively. This activity was completely blocked by the dominant negative mutants RAS17N or MEKAA, suggesting that p70 functions upstream of RAS. Unlike wild-type p70, expression of p70-DeltaC1 or p70-DeltaC2 mutant did not interfere with the ability of ST5-p126 to stimulate ERK2. Taken together, the data suggest that the COOH-terminal tail, residues 489-609, contains some of the critical determinants for the function of p70. Loss of this region converts the protein from an inhibitor to a constitutive activator of the RAS-ERK2 pathway.

Formation of a covalent complex between the 80,000-dalton adenovirus terminal protein and 5'-dCMP in vitro.

An in vitro adenovirus DNA replication system catalyzed the formation of a covalent complex between an 80,000-dalton protein and 5'-dCMP in the presence of [alpha-32P-dCTP, MgCl2, ATP, and adenovirus (Ad) DNA with a protein covalently bound to the 5' end of each strand (Ad DNA-prot). The requirement for Ad DNA-prot in this reaction was similar to that for in vitro DNA replication. When dATP, dTTP, and the 2',3'-dideoxynucleoside triphosphate (ddNTP) ddGTP were included in the reaction mixture, an elongated complex was detected, which consisted of an 80,000-dalton protein bound to a 26-base oligonucleotide. Formation of the elongated product, but not of the protein-dCMP complex, was inhibited by ddATP, ddCTP, or ddTTP. The requirements for formation of the protein-dCMP complex, the nature of the linkage between protein and dCMP, the size of the protein, and the existence of elongated forms indicated that the protein associated with the complex was identical to the 80,000-dalton Ad terminal protein found on replicating DNA molecules as described by Challberg et al. [Challberg, M. D., Desiderio, S. V. & Kelly, T. J., Jr. (1980) Proc. Natl. Acad. Sci. USA 77, 5105-5109].

Activation of extracellular signal-regulated kinase 2 by a novel Abl-binding protein, ST5.

The human ST5 gene encodes three proteins with predicted molecular masses of 126, 82, and 70 kDa. These widely expressed proteins share a C-terminal region that bears significant sequence homology to a group of GDP/GTP exchange proteins for the Rab3 family of small GTP binding proteins. The N-terminal region of the largest ST5 protein, p126, contains two proline-rich sequences, PR1 and PR2, with consensus motifs similar to Src homology 3 (SH3) binding regions and to mitogen-activated protein kinase (MAPK) phosphorylation sites. Based on these properties, we sought to investigate the activity of ST5 proteins in signal transduction pathways. In vitro, p126 displayed preferential binding to c-Abl SH3, as compared with other SH3 domains. This interaction was mediated by the PR2 sequence. In vivo, expression of p126, but not p82 or p70, activated MAPK/ERK2 in response to EGF in COS-7 cells. Expression of c-Abl with p126 greatly enhanced this activity. Deletion of PR1 blocked the ability of p126 to activate ERK2. Deletion of PR2 did not affect the basal activity, but blocked the stimulatory effect of c-Abl. Whereas p82 expression had no effect on ERK2 activation by p126, p70 completely abrogated this activity. These observations suggest that ST5 can function as a signaling protein and can provide a link between c-Abl and ERK2.

Loss of heterozygosity in fibrocystic change of the breast: genetic relationship between benign proliferative lesions and associated carcinomas.

Loss of heterozygosity (LOH), a genetic change frequently detected in cancer, can also occur in benign epithelial foci in the breast. To characterize LOH in benign breast tissue, 32 cases containing the various components of fibrocystic change in the absence of malignancy were studied. Microdissected foci of ductal hyperplasia, apocrine metaplasia, sclerosing adenosis, and morphologically normal terminal duct lobular units (TDLUs) were analyzed for LOH at 14 polymorphic loci representing seven chromosomal arms. LOH was detected in 22% of normal TDLUs (6/27), 17% of adenosis (4/23), 19% of hyperplasia (4/21), and 53% of apocrine metaplasia (10/19) specimens. Because of the high percentage of LOH in apocrine metaplasia in nonneoplastic specimens, the genetic relationship between apocrine metaplasia and cancer was studied in a panel of breast cancer cases. Of 14 examples of apocrine metaplasia adjacent to a carcinoma, seven were found to have LOH with at least one marker. In all seven cases, the tumor and apocrine metaplasia shared LOH at one or more markers. The results demonstrate that LOH occurs frequently in the components of fibrocystic change as well as in normal TDLUs and suggest that foci of apocrine metaplasia can share a genetically altered precursor cell with an associated carcinoma.

Human herpesvirus 8 genome is not found in whole bone marrow core biopsy specimens of patients with plasma cell dyscrasias.

Recently, molecular evidence of the gamma herpesvirus, human herpesvirus 8 (HHV-8), was found in the nonmalignant bone marrow stromal cells of patients with multiple myeloma using a polymerase chain reaction (PCR)-based assay. Other investigators have been unable to confirm either the presence of HHV-8 using molecular techniques or serologic evidence of prior infection with HHV-8. In order to maximize the likelihood of detection of small quantities of the virus and minimize the risk of potential nucleic acid contamination, we used entire bone marrow biopsy core specimens for DNA extraction and amplification. These specimens included both malignant plasma cells and bone marrow stromal cells and were subjected to minimal manipulation prior to DNA extraction and PCR. We tested eight patients with various plasma cell dyscrasias and compared them to negative controls with non-Hodgkin's lymphoma using standard PCR assays utilizing the KS330(233)primers and probe for HHV-8. This assay is reproducibly positive in Kaposi's sarcoma tissue. We found no evidence of HHV-8 DNA in either the lymphoma controls or the samples from patients with the plasma cell dyscrasias using these methods. We conclude that HHV-8 is unlikely to play a major role in the pathogenesis of the plasma cell dyscrasias in the majority of patients with these diseases. This report adds to the body of evidence that HHV-8 is not associated with plasma cell dyscrasias like multiple myeloma.

Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form.

The 80,000-dalton form of the adenovirus (Ad) terminal protein (pTP) has been purified from Ad-infected HeLa cells. pTP was assayed by its ability to form a covalent complex with dCMP. The protein copurified with an activity that is essential for in vitro Ad DNA replication (Ad protein activity) as well as with a DNA polymerase activity that was distinguished from those of HeLa cell DNA polymerases alpha, beta, and gamma. The Ad protein-associated DNA polymerase activity was detected with activated DNA but not with poly(rA).oligo(dT) as template and was insensitive to aphidicolin and sensitive to N-ethylmaleimide. The Ad protein, DNA polymerase, and pTP-dCMP complex-forming activities sedimented in a glycerol gradient as a single peak with an apparent molecular size of 180,000 daltons. NaDodSO4/polyacrylamide gel analysis of the glycerol gradient fraction showed major bands of 80,000 and 140,000 daltons. The 80,000-dalton band was identified as pTP by comparison of its tryptic peptide map with that of the 55,000-dalton form of the terminal protein, which was purified from Ad virions.

Adenovirus DNA replication in vitro: identification of a host factor that stimulates synthesis of the preterminal protein-dCMP complex.

A protein factor that participates in the formation of a covalent complex between the 80,000-dalton precursor of the adenovirus (Ad) terminal protein (pTP) and 5'-dCMP has been isolated and characterized. This 47,000-dalton protein, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor I. It is free of detectable DNA polymerase alpha, beta, and gamma activities. In the presence of Ad DNA-prot, the Ad-protein fraction (containing the pTP and the Ad-associated DNA polymerase), ATP, Mg2+, and dCTP, nuclear factor I stimulates formation of the pTP-dCMP complex. Addition of the Ad DNA binding protein (Ad DBP) renders the formation of the pTP-dCMP complex completely dependent on the addition of nuclear factor I. When Ad DNA-prot is replaced with phi X174 single-stranded circular DNA, pTP-dCMP complex formation requires only the Ad-protein fraction; Ad DBP and ATP are inhibitory and nuclear factor I has no effect on this reaction. This suggests that the initiation reaction observed with Ad DNA-prot in the absence of Ad DBP occurs at single-stranded DNA sites. In the presence of Ad DBP, these sites are blocked thus creating a requirement for nuclear factor I in pTP-dCMP complex formation.

Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication.

A complex containing the 80,000-dalton precursor to the adenovirus (Ad)-encoded terminal protein (pTP) and a 140,000-dalton protein is required for Ad DNA replication in vitro. This complex has been separated into subunits by glycerol gradient centrifugation in the presence of urea. The isolated 140,000-dalton subunit contains a DNA polymerase activity which can be differentiated from all host DNA polymerases. No enzyme activity was detected with the isolated pTP. The requirements for reactions involved in the initiation of Ad DNA replication were determined by using the isolated subunits. The covalent addition of dCMP, the first nucleotide in the DNA chain, to the pTP, which serves as the primer for replication, required the DNA polymerase subunit as well as the pTP. Synthesis of viral DNA in vitro also required both subunits. The properties of the DNA polymerase suggest that it may be a viral gene product.

Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149.

The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis in vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982) J. Biol. Chem. 257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982) J. Biol. Chem. 257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.

Differential expression of the human ST5 gene in HeLa-fibroblast hybrid cell lines mediated by YY1: evidence that YY1 plays a part in tumor suppression.

Through a mutational analysis of a differentially regulated enhancer, we present evidence that supports a role for the transcription factor YY1 in tumor suppression in HeLa/fibroblast somatic cell hybrids. The human ST5 gene was previously shown to be expressed as three RNA species, 4.6, 3.1 and 2.8 kb in length. Whereas the two larger species are expressed at similar levels in all cell lines examined, the 2.8 kb mRNA is expressed specifically in non-tumorigenic hybrids. In this study, the basis for the differential expression of this mRNA species was investigated. The message was shown to originate from a promoter located within an intron of the ST5 gene. An enhancer located approximaely 1500 nt upstream of the start site was required for cell type specific expression. Mutational analysis of this enhancer revealed an AP1 site and five YY1 sites which were necessary for full enhancer activity. Levels of YY1 DNA binding activity were found to be as much as 6-fold higher in the non-tumorigenic cells relative to the tumorigenic cells, while AP1 activity was similar in both cell types. These results suggest that a signaling pathway targeting YY1 may play an important role in tumor suppression in HeLa-fibroblast hybrids.

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.

Genetic heterogeneity in ductal carcinoma of the breast.

Genetic heterogeneity in breast cancer has been observed both by cytogenetic and loss of heterozygosity (LOH) analyses; however, the frequency with which genetically heterogeneous clones arise is unknown. In this study, a panel of 115 breast carcinomas was analyzed to determine the extent of clonal divergence in tumor foci at progressive stages of tumor evolution. Intraductal, infiltrating, and metastatic tumor components were microdissected from each tumor and tested for LOH at 20 microsatellite markers on seven chromosomal arms. Of these cases, 24 (21%) demonstrated genetically divergent clones during tumor progression. Clonal divergence, inferred from discordant LOH patterns, was observed most commonly between intraductal and infiltrating tumor (18 cases), but was also demonstrated between infiltrating and metastatic tumor (11 cases). Discordant LOH was observed with markers on one chromosomal arm in 16 cases, on two in 7 cases, and on four in 1 case, and was observed most commonly with markers on 17p, 17q, and 16q. More detailed microdissection of four cases provided evidence for a specific chronology of genetic alterations occurring during the progression of each tumor. The results indicate that the different tumor components observed microscopically in breast cancer specimens often represent genetically divergent clones.

Rapidly progressive T cell lymphoma presenting as acute renal failure: case report and review of the literature.

We describe a case of peripheral T cell lymphoma that is remarkable for its fulminate course and selective targeting of both kidneys. The patient was a 6-year-old girl who was in her usual state of good health until the onset of abdominal pain and fever. She was treated for acute oliguric renal failure and visual disturbances. A renal biopsy was performed. Biopsy findings were interpreted as suggestive of a vasculitic process, and treatment was initiated for a presumptive diagnosis of Wegener's granulomatosis. The patient died 3 days following admission, and autopsy revealed extensive bilateral kidney infiltration by a peripheral T cell lymphoma. The remainder of the body was spared with the exception of mild infiltration of the pulmonary parenchyma and choroid plexus by neoplastic lymphocytes. The neoplastic nature of the disease was confirmed utilizing immunoperoxidase stains and T cell receptor gene rearrangement. Primary renal lymphoma and renal failure attributable to involvement by lymphoma are rare findings that should be considered when other more common causes of renal insufficiency have been excluded. The presenting clinical complaints are generally of short duration, nonspecific, and atypical. Most patients exhibit oliguria. Physical examination may reveal hepatosplenomegaly, lymphadenopathy, and flank and/or abdominal mass(es). Laboratory findings frequently include an elevated serum creatinine, blood urea nitrogen, lactate dehydrogenase, and a mild proteinuria. Electrolyte abnormalities are variably present. Possible radiographic findings include hypodense or hypoechoic renal lesions and diffuse bilateral renal enlargement. Although the prognosis is dismal, survival may be prolonged utilizing current treatment modalities, and rare patients may be "cured" of disease. The clinical presentation, radiological findings, and prognosis of patients with clinically evident renal involvement by non-Hodgkin's lymphoma are discussed.