Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing [32Plorthophosphate and 10% (vol/vol) fetal bovine serum. A 32P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The 32p labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The 32p label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as [32PJAMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the 32P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and[32P]NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When 32P-labeled cultures were incubated in medium containing unlabeled phosphate, the 32p label was lost from the EF-2 within 30 min. Diphtheria toxin and Pseudomonas toxin A specifically ADP-ribosylate elongation factor-2 (EF-2) and thereby block protein synthesis (1, 2). The specificity of the reaction resides in the unique ADP-ribose acceptor site in EF-2 that is a posttranslationally modified histidine residue, called diphthamide (3, 4). Conservation of the diphthamide residue throughout eukaryotic evolution (5-7) suggests that it serves an essential function in cellular metabolism and is not present solely for the purpose of bacterial intoxication of cells. This view is supported by the discovery of an ADP-ribosyltransferase that co-purifies with EF-2 from hamster cells (8). This enzyme appears to modify the same diphthamide residue of EF-2 modified by diphtheria toxin and Pseudomonas toxin A. Similar enzyme activities were subsequently found in extracts of several other cell types (9-11). Thus far, characterization of the enzyme has been limited to in vitro reactions.To demonstrate the transferase activity in cells, we now have isolated and characterized its unique reaction product, ADPribosylated EF-2, from polyoma virus-transformed baby hamster kidney (pyBHK) cells. The ADP-ribosylation of EF-2 in cells is limited to a fraction of the EF-2 and responds to the serum concentration of the culture medium. MATERIALS AND METHODSLabeling pyBHK Cells. Cells were grown in Eagle's minimal essential medium with Earle's salts (MEM) as described (12). Cultures were established in MEM containing 10% (vol/vol) fetal bovine serum (FBS) by seeding 60-mm culture dishes with 5 x 104 or 2.5 x 104 cells for subsequent labeling in medium containing 2% or 10% dialyzed FBS, respectively. At the time of lysis, both 2% and 10% FBS-treated cultures contained 9 X ...

Anti-[ADP-ribosylated elongation factor 2 (EF-2)] antiserum has been used to immunoprecipitate the modified form of EF-2 from polyoma-virus-transformed baby hamster kidney (pyBHK) cells [Fendrick, J. L. & Iglewski, W. J. (1989) Proc. Nut1 Acad. Sci. USA 86, 554-5571. This antiserum also immunoprecipitates a 32P-labelled protein of similar size to EF-2 from a variety of primary and continuous cell lines derived from many species of animals. One of these cell lines, Chinese hamster ovary CHO-K1 cells was further characterized. The time course of labelling of ADP-ribosylated EF-2 with [32P]orthophosphate was similar in pyBHK cells and in CHO-K1 cells. The kinetics of labelling were more rapid for cells cultured in 2"/0 serum than 10% serum, with incorporation of 32P reaching a maximum at 6 h and 10 h, respectively. EF-2 mutants of pyBHK and CHO-K1 cells resistant to diphtheria-toxin-catalyzed ADP-ribosylation of EF-2 remain sensitive to cellular ADP-ribosylation of EF-2. The 32P-labelled moiety of ADP-ribosylated EF-2 was digested by snake venom phosphodiesterase and the product was identified as AMP. The same 32P-labelled tryptic peptide was modified by toxin in wild-type EF-2 and by the cellular transferase in mutant EF-2. When purified EF-2 from pyBHK cells was incubated with [c~rhonyl-'~C]nicotinamide and diphtheria toxin fragment A, under conditions for reversal of the ADP-ribosylation reaction, [ 14C]NAD was generated. The results suggest that cellular ADP-ribosylated EF-2 exists in a variety of cell types, and the ribosylated product is identical to that produced by toxin ADP-ribosylation of EF-2, except in diphthamide mutant cells. Studies with the mutant cell lines indicate that the toxin and the cellular transferasc, however, recognize different determinants at the ADP-ribose acceptor site in EF-2. The cellular transferase does not require the diphthamide modification of the histidine ring in the amino acid sequence of EF-2 for the transfer of ADP-ribose to the ring. Therefore, we would expect the cellular transferase active site to be similar to, but not identical to, the critical amino acids demonstrated in the active site of diphtheria toxin and Pseudoomonus exotoxin A. Eukaryotic ADP-ribosyltransferases, which transfer the ,4DP-ribose moiety from NAD to elongation factor 2 (EF-2) in in v i m reactions, have been isolated from polyoma-virustransformed baby hamster kidney (pyBHK) cells and from bovine liver [ I , 21. Subsequently, similar enzymes were isolated from rabbit reticulocytes [3] and rat liver [4]. These eukaryotic enzymes are intriguing since they modify a site in the same Correspondcnw to W. J . Iglewski,

Ovarian steroid hormones play a critical role in regulating mammary gland growth and development. The mammary gland sequentially acquires and cyclically exhibits proliferative responses to estrogen and/or progesterone from birth to postmenopause. The focus of this review is to present our current understanding of estrogen and progesterone receptor distribution in epithelial and stromal cells and their functions in relation to mammary gland development. Insights gained from the study of the normal mammary gland are relevant to our understanding of the conditions which may predispose women to the development of breast cancer as well as to alterations in hormonal regulation that occur in breast cancer.

The purpose of the present study was to investigate the role of extracellular matrix proteins (ECMs; collagens I and IV, fibronectin, and laminin) in modulating proliferative responses of normal mammary epithelial cells in serum-free culture to epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I). As EGF and IGF-I can alter steroid responses, the interactions among growth factors, estrogen, and R5020 were also investigated. We report the novel finding that all ECMs tested, but not a nonspecific attachment factor, poly-L-lysine (PL), promoted a highly synergistic proliferative response to EGF plus IGF-I. EGF receptors were significantly increased with culture time on all ECMs, but not on PL. IGF receptor expression was significantly 2- to 4-fold higher on all ECMs compared with PL. EGF decreased IGF-binding protein-2 (IGFBP-2) and IGFBP-3 by more than 50% in the presence of IGF-I on PL or collagen I. These results indicate that ECM-specific IGF-I/EGF synergism occurs in response to ECM up-regulation of growth factor receptors and EGF down-regulation of inhibitory IGFBPs. Growth factors did not synergize with estrogen and/or R5020. Instead, estrogen plus R5020 decreased EGF-plus IGF-I-induced proliferation in an ECM-dependent manner. These studies demonstrate that proliferation of normal mammary epithelial cells involves complex interactions among steroids, growth factors, binding proteins, and ECMs.

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Enhancement of plaque formation by IHN virus on CHSE-214 cells was obtained with 2-5 /(g/ml polyhrene. The enhancement is due to increased infectivity of small plaque variants in the IHN virus stock.

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Five fish cell lines (CHSE-214, STE-I37, RTG-2, EPC and FHM) were compared for sensitivity to infeetious haematopoietic necrosis virus (IHNV) from samples obtained from naturally-infected fish. Infeetious ovarian fluids were obtained from steelhead trout, Salmo gairdneri Riehardson, at the Round Butte Hatchery in central Oregon and tissue homogenates were prepared from chinook salmon, Oncorhynchus tshawytscha (Walbaum), alevins during an IHN virus epizootic at the Elk River Hatchery in coastal Oregon. The only lines to show characteristic viral cytopathology by plaque or end-point dilution assay for the steelhead trout virus isolate were the EPC and FHM cell lines. The chinook salmon isolates produced CPE in CHSE-214, STE-137, FHM and EPC cells. The titre of the salmon virus isolate was 10-50-fold higher on FHM and EPC cells by both assay methods. Neither by end-point nor plaque assay did the Round Butte or Elk River isolates produce CPE on RTG-2 cells. With both virus isolants both cell lines showed that greater sensitivity was obtained with plaque assay than with end-point titration. Pre-treatment of the cells with the polyeation. polybrene, did not increase the virus titre in either assay. However, a transient enhancement in virus titre was observed in polybrene-treated STE-137 and CHSE-214 cells.