Brenda I. Gerwin

ErbB-2 kinase is required for constitutive stat 3 activation in malignant human lung epithelial cells.

Overexpression of the growth factor receptor ErbB‐2/Her2/Neu has been implicated in the development of non‐small‐cell lung cancer. We have reported that the transformation of human lung epithelial cells by c‐erbB‐2 also requires an active ErbB‐1 (EGF receptor) and the autocrine production of its ligand, TGF‐α. In this report, we demonstrate that STAT 3 is constitutively activated in these cells by the TGF‐α–stimulated ErbB‐1/‐2 heterodimer complex. STAT 3 activation was confirmed by mobility shift assays and nuclear localization. ErbB‐1 was required, but not sufficient for the TGF‐α–induced activation of STATs. Inhibition of ErbB‐2 kinase activity by tyrphostin AG825 prevented the constitutive activation of STAT 3 in the TGF‐α–producing, ErbB‐1 expressing cell line. Our results demonstrate a requirement for ErbB‐2 kinase activity to establish constitutive STAT 3 activation resulting from an autocrine ErbB‐1/ TGF‐α loop. Int. J. Cancer 83:564–570, 1999. Published 1999 Wiley‐Liss, Inc.

Dissimilar peptide growth factors can induce normal human mesothelial cell multiplication

SummaryQuiescent normal human mesothelial (NHM) cells will undergo one round of DNA synthesis when they are incubated in a defined medium consisting of LHC basal medium supplemented with hydrocortisone, insulin, transferrin, and one of the following peptide mitogens: epidermal growth factor; transforming growth factor beta (1 or 2); platelet derived growth factor (a,b heterodimer or b,b homodimer); fibroblast growth factor (acid or basic forms); interleukin 1 (alpha or beta forms); interleukin 2; interferon gamma; interferon beta; or cholera toxin. However, sustained cell multiplication does not occur unless the medium contains hydrocortisone, insulin, transferrin, any one of the above-listed peptide growth factors and high density lipoproteins. Growth can be increased twofold if the medium contains certain combinations of these mitogens and high density lipoproteins. The finding that NHM cells can respond to a broad spectrum of growth factors supports the possibility that an autocrine mechanism may be part of the mechanism that leads to transformation of these cells by asbestos.

Phenotypic mixing between N- and B-tropic murine leukemia viruses: Infectious particles with dual sensitivity to Fv-1 restriction

In effort to understand how N or B tropism is determined in murine leukemia virus (MuLV) particles, we analyzed the MuLV produced after dual infection of mouse cells by N- and B-tropic MuLV. The progeny MuLV from such a mixed infection are sensitive to Fv-1 restriction in both N- and B-type cells, but are still highly infectious for mouse cells which do not exhibit Fv-1 restriction. This dual sensitivity to Fv-1 restriction is a phenotypic property of MuLV produced by mixedly infected cells, since individual virus clones derived from this MuLV are either N- or B-tropic. In further experiments, we superinfected murine sarcoma virus (MSV)-transformed cells with mixtures of N- and B-tropic MuLVs. The rescued MSV is restricted in its ability to transforms both N- and B-type cells. The results suggest that N- and B-tropic MuLVs specify different determinants, which are incorporated into virions along with the viral genome and which are the recognition sites for Fv-1 restriction. The presence of a given determinant in a virion renders the virus sensitive to restriction in cells of the opposite Fv-1 type.

Isolation of a continuous epithelioid cell line, HBT-3, from a human breast carcinoma.

Summary A continuous line of human breast carcinoma cells, HBT-3, was established in culture following collagenase treatment of a mucus producing adenocarcinoma. The cells are epithelioid in appearance, multiply rapidly, have a cloning efficiency of approximately 70%, and exhibit an abnormal karyotype with a mode of 66-69 chromosomes/cell and 3 markers. Tumor material and pathological diagnosis were provided through the courtesy of Dr. J. D. Mashburn, Director of Laboratories, Washington Sanitarium and Hospital, Takoma Park, MD. Dr. C. S. Stulberg, Senior Research Associate, The Child Research Center of Michigan, Detroit, kindly performed the immunofluorescence cell typing tests. A portion of these studies was carried out at Bionetics Research Laboratories, Inc., Bethesda, MD, under contract 69-2160 from the Special Virus Cancer Program of the National Cancer Institute. Technical assistance of Cathy K. Smith, Nancy Tuttle Fuller, and Diane Robertson is gratefully acknowledged. The authors thank Drs. Peter J. Fischinger and Tadao Aoki of the National Cancer Institute for their advice and many helpful discussions.

Two active forms of RD-114 virus DNA polymerase in infected cells.

Two forms of DNA polymerase are present in RD-114-infected human, dog, and mink cells, but are not detectable in uninfected cells. The two enzymes are indistinguishable catalytically and immunologically, but differ with respect to molecular weight and elution position from (dT)12-18-cellulose and phosphocellulose. The large enzyme (equivalent 95,000 daltons) is found in the infected cells, but not the virions produced by these cells. The virions contain only the smaller enzyme (equivalent 70,000 daltons). The larger form may represent a mammalian viral equivalent to the beta subunit of avian RNA tumor virus DNA polymerase.

Murine sarcoma virus defectiveness. Viral polymerase expression in murine and nonmurine host cells transformed by S + L - type murine sarcoma virus.

Sarcoma virus-positive, leukemia virus-negative (S + L -) mouse cells transformed by defective but rescuable murine sarcoma virus (MSV) release noninfectious type C virions with a quantitative deficiency of viral-type RNA-dependent DNA polymerase (RDDP). Human S + L - cells containing the same genome fail to express any detectable viral-type reverse transcriptase. To study this difference in MSV expression, second-generation MSV from the S + L - human cells is recloned in dog, mink, and back into mouse cells. Heterologous host dog and mink S + L - cell clones, like human S + L - cell clones, fail to release viral-type reverse transcriptase into culture supernatant fractions. All second-generation homologous mouse S + L - cell clones again release viral reverse transcriptase in supernatant fractions, indicating that the MSV genome has not been altered in this function. Using (dT)(12-18)-cellulose and phosphocellulose chromatography of cellular polymerase preparations, no intracellular buildup of unreleased murine reverse transcriptase is detected in the S + L - clones. The data presented here suggest that the MSV genome is defective in the information for the viral core protein RDDP. The implications of these findings for the genetic study of MSV are discussed.

Murine sarcoma virus defectiveness: Serological detection of only helper virus reverse transcriptase in sarcoma virus rescued from nonmurine S+L− cells

Abstract Murine sarcoma virus (MSV) defectiveness was studied by examining MSV rescued by RD-114 virus superinfection of both dog and human heterologous host cells nonproductively transformed by the S+L− strain of Moloney MSV (S+L− cells). Biological assays showed that the rescued MSV pseudotype was present in excess over the RD-114 helper virus. The biologically determined virus ratio corresponded to that determined by virus nucleic acid hybridizations. Antisera distinguishing the DNA polymerase of murine type C virus from that of RD-114 virus demonstrated that the only detectable RNA-dependent DNA polymerase present in the rescued MSV virions was that of RD-114 helper virus. No murine virus polymerase was detectable within the virions. Previous data have demonstrated that no murine-viral polymerase is associated with S+L− heterologous human, dog, and mink host cells. The work presented here demonstrates that cells producing infectious MSV and helper virions still do not express murine polymerase within released MSV virions. These findings suggest that heterologous host-cell control mechanisms are not preventing MSV polymerase expression in these cells. Instead, the rescued MSV virions acquire the polymerase protein from the rescuing helper virus. These data provide evidence that the MSV genome lacks the information necessary for polymerase, and requires for the production of infectious progeny the functioning of a helper virus replication gene set supplying at least viral envelope antigen(s) and virion core protein polymerase.

Revertants of mouse cells transformed by murine sarcoma virus: II. Flat variants induced by fluorodeoxyuridine and colcemid

Abstract Mouse sarcoma virus-transformed 3T3FL cells (S+L− cells) produced spontaneously and at variable rates flat variants with some properties of nontransformed cells. The frequency of occurrence of such variant cells increased after treatment of S+L−cells with fluorodeoxyuridine (FdUrd) or Colcemid. In one S+L− subline (3–360), the spontaneous flat variants (S) occurred at the rate of about 1 in 80 clonal colonies, and, after treatment with Colcemid or FdUrd, flat variants were observed in 1 of 20 and 30 colonies, respectively. In another S+L− subline (3–321), S variants normally occurred less than 1 in 1000 colonies, and the same treatments increased the frequency to approximately 1 in 70 and 55 colonies, respectively. Both the FdUrd and Colcemid-induced flat variants (F and C) resembled 3T3FL cells morphologically, grew to low saturation densities, and exhibited cloning efficiencies in soft agar which were 10 −1 to 10 −4 times lower than that of S+L− cells. However, except for one S subline, they contained murine leukemia group-specific antigen (s) without demonstrable virus production and reverse transcriptase activity. Murine sarcoma virus (MSV) was no longer rescuable by superinfection with murine leukemia virus (MuLV) or by cell fusion with 3T3PL, BALB/3T3, or normal rat kidney cells. All flat variant cells were susceptible to MSV and MuLV infection, and some degree of enhancement of sensitivity to MSV and MuLV infection was observed in most variant cultures. All flat variant sublines possessed the property of agglutinability by concanavalin A as high as that of S+L− cells. Some flat variant clones spontaneously underwent retrans-formation during extended cultivation. In contrast to previously described S+L−cell revertants, which spontaneously retransformed morphologically without a rescuable MSV genome, in the present experiments retransformation with a rescuable MSV genome was observed in 1 of 4 S sublines and 2 of 5 F sublines, but in none of 4 C sublines. Furthermore, 1 of 5 F sublines gave rise to a clone which was flat but which otherwise resembled S+L− cells. In the S and C variants, the loss of expression of transformation was associated with an increase in chromosome number. However, the chromosome number of F variants was similar to or slightly less than that of parental S+L− cells. These studies demonstrated that the reversion of S+L− cells to flat variants occurred by different mechanisms apparently involving either the viral or cellular genes or both, and that some flat variant sublines had retained at least one complete MSV genome in nondetectable form.

Functional analysis of reverse transcription by a frameshift pol mutant of murine leukemia virus

Endogenous reverse transcription by wild-type murine leukemia virus (MuLV) was compared to that catalyzed by clone 23, a pol mutant containing a reverse transcriptase protein which lacks the carboxyl-terminal third of the molecule (J. G. Levin, S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin (1984), J. Virol. 51, 470-478). Competition immunoassays revealed that mutant virions contain normal amounts of polymerase protein, indicating that the lack of carboxyl-terminal sequences does not alter normal processing of enzyme precursors. Although the mutant enzyme was previously shown to have the ability to copy and degrade RNA:DNA hybrids, the present study demonstrates that it is defective in functions required to generate full-length copies of viral DNA. Analysis of products of endogenous reverse transcription showed that minus-strand strong-stop DNA is formed and that mutant virions synthesize a series of minus-strand DNA intermediates up to 2.2 kb in length. Comparison of mutant and wild-type MuLV reaction products indicated that the 2.2-kb termination site of the mutant corresponds to a normal pausing region for the wild-type enzyme. Computer analysis of sequences and structure within pausing regions suggested the involvement of C-rich consensus sequences plus multibranch loop structures in the general phenomenon of enzyme-pausing during reverse transcription.

Cultured Human Mesothelial Cells are Selectively Sensitive to Cell Killing by Asbestos and Related Fibers: A Potential in Vitro Assay for Carcinogenicity

The use of man-made fibers as substitutes for asbestos is increasing, and it is important to determine the potential health hazards, including potential carcinogenicity, of these fibers. It is of particular interest to develop short-term in vitro assays that could significantly decrease the need for animal testing as well as provide rapid and reliable information to industry during the time that applications for new fibers are being developed. One potential rapid screening assay for mesothelioma-causing fibers is selective cytotoxicity for cultured human mesothelial cells. Cell killing of cultured normal human mesothelial cells by amosite, chrysotile, or crocidolite fibers occurs at levels of exposure approximately 50 fold less those required to produce cell killing cultured human lung fibroblasts. This differential cytotoxic effect is also observed for erionite, a fibrous zeolite linked to mesothelioma in Turkey, and Code 100 glass fiber found to cause mesothelioma in laboratory animals. In contrast, both mesothelial cells and fibroblasts require similar high levels of exposure to fibers such as Wollastonite, glass wool, and refractory ceramic fibers for cell killing. Non-fibrous particulates such as aluminum oxide, and a variety of chemicals are also toxic for both cell types at similar concentrations. Thus, human mesothelial cells, a selective in vivo target to the carcinogenic effects of asbestiform fibers, are also a selective in vitro target of the cytotoxic effects of these fibers. Differential cytotoxicity for cultured mesothelial cells and fibroblasts may be useful for predicting the potential of new man-made fibers to cause mesothelioma.