Cathepsin B and complement C3 are major comigrants in the estrogen-induced peroxidase fraction of rat uterine fluid.

The luminal fluid of estrogen or DES-stimulated uterus of immature rats contains 10-12 isoforms of peroxidase between pI 4.5-6.0. N-terminal amino acid sequencing of diaminobenzidine-peroxidase bands eluted from IEF and SDS-PAGE gels showed the presence of cathepsin B and the complement family of proteins as the major comigrants. Sequential treatment of uterine fluid by cation, anion, and size exclusion chromatography resulted in a five-fold purification of peroxidase having a specific activity of 273 units/mg. Mass spectrometric studies of bands isolated from SDS-PAGE gels from the size-exclusion purified peroxidase fraction showed the presence of complement C3 along with novel previously uncharacterized proteins. Two dimensional electrophoresis followed by N-terminal amino acid sequencing confirmed the presence of cathepsin B isoforms and isoforms of a novel protein at approximately 87 kDa. Identification by mass spectrometry from the database for this novel protein was inconclusive but could most likely be a candidate for estrogen-induced peroxidase. Results conclusively prove that cathepsin B and complement C3 are major proteins in the estrogen-induced peroxidase fraction of uterine fluid.

Fibronectin/fibroblast growth factor/cell matrix signaling pathways and reciprocal membrane interaction may be the regulators of cell growth and apoptosis in Trypanosoma musculi in co-cultures with fibroblasts.

Trypanosoma musculi cultivated in medium containing serum alone or in the absence of fibroblasts in vitro were transformed into rounded, immotile cells, incapable of division and infectivity. Only in close contact with fibroblasts could the parasites survive and grow indefinitely. This report established the identity of the splenic 'sustentacular' cells as fibroblasts and utilized immunocytochemistry to demonstrate the putative cytoskeletal and membrane-associated molecules that may be involved in the control of growth and division, and apoptosis of T. musculi in vitro. The results indicated that cells that reacted intensely for fibroblast growth factor (FGF) also displayed a complex cytoskeletal system of F-actin bands underlying the plasma membrane of the fibroblast cell body and its numerous processes. Among the cytoskeletal and membrane glycoproteins, fibronectin, I-CAM, laminin, occludin, vinculin and desmin were most prominent. Fibronectin was most highly enhanced on the cell membrane and deposited as 'finger prints or tracks' on the extracellular culture surfaces. Transmission and scanning electron microscopy confirmed the intimate contact between trypanosomes and fibroblasts, however, neither membrane fusion or junctions were apparent. Our results suggested that a fibroblast-derived, membrane-associated factor appeared to be the putative growth regulator and apoptosis inhibitor in co-cultures of spleen-derived fibroblasts and T. musculi.

Estrogen receptor, growth factor receptor and protooncogene protein activities and possible signal transduction crosstalk in estrogen dependent and independent breast cancer cell lines.

Binding of estrogen to its receptor (ER) activates early genes that drive responsive cells through the proliferative phase. Earlier studies to evaluate the expression of protooncogenes, growth factors, growth factor receptor and steroid hormone receptor gene activities in the rat uterine system indicated complex pathways that involve significant 'crosstalk' between ER-systems and signal transduction pathways (Bhattacharyya et al., 1994). To analyze the interactions between these factors, we examined two well characterized estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. Antibodies to estrogen receptor, epidermal growth factor receptor, c-Fos, c-Jun, and Ras proteins, protein kinases involved in receptor tyrosine kinase signal transduction pathway, MEK1 and phosphotyrosine were utilized in immunocytochemical localization experiments to evaluate temporal expression of these factors in response to estrogen treatment. ER, which was diminished in MCF-7 cells grown in estrogen-stripped medium, increased 9-fold in estrogen-reconstituted medium by 120 min. Fos and Jun appeared at nuclear and perinuclear cytoplasmic sites within 60 min after estrogen treatment in MCF-7 cells. Fos/Jun proteins were prominent in MDA-MB-231 cells, especially in association with actin filaments. Immunolabeling studies revealed no EGF-r in MCF-7 cells, while MDA-MB-231 cells contained intense EGF-r labeling in the plasma membrane. Ras protein was prominent in the cytoplasm and at the cell surface within 60 min after treatment of MCF-7 cells with estrogen. Ras was intense in MDA cells. Similarly, MCF-7 and MDA cells contained high concentrations of MEK1 and phosphotyrosine (pTyr) containing proteins in their cytoplasm and immunolabeling remained high as long as MCF-7 cells were grown in medium containing estrogen. It is speculated that MEK1 (cytoplasmic) functioning through Fos/Jun or Myc/Max (nuclear) may regulate the activity of AP-1 transcription factor. In all cases however, MEK1 and pTyr protein labeling was more intense in the highly metastatic and hormone independent MDA-MB-231 breast cancer cells. Results revealed signal transduction pathway proteins in ER+ estrogen dependent cells suggesting possible crosstalk between both receptor pathways during the proliferative phase of MCF-7 cells.

Protooncogene, growth factor, growth factor receptor, and estrogen and progesterone receptor gene expression in the immature rat uterus after treatment with estrogen and tamoxifen.

The mechanism(s) by which estrogen regulates cell growth in target cells and the cascade of biochemical changes associated with growth have not yet been fully determined. Equally undetermined is an understanding of the mechanism(s) by which tamoxifen blocks estrogen-regulated growth. This study, therefore, attempts to define and correlate the physiological processes in the rat uterus following estrogen and tamoxifen administration with temporal events manifested by mRNA expression of protooncogenes (m-myc, c-ras, c-fos, c-jun), growth factors and/or inhibins (IGF-1, IGF-2, IGF-2 Exon 1 and Exon 2), EGF, TGFB-1, -2, -3), growth factor or inhibin receptors (EGFr, TGFB-2r, TGFB-3r), and estrogen-induced differentiative proteins including estrogen receptor (ER), progesterone receptor (PR). In this study, mRNA was isolated from hormone and antagonist-treated rat uteri at 0', 15', 30', 1 h, 6 h, 24 h, 48 h and 72 h. Expression studies were analysed by dot/Northern blot hybridization with cDNA or oligonucleotide probes for the RNAs mentioned above. Nuclear runoff transcriptional assays were also performed. Our data suggest that fos, myc, ras and jun protooncogenes were expressed from 15' to 48 h after treatment with either estrogen or tamoxifen. Tamoxifen treatment resulted in diminished expression, but incomplete inhibition of the protooncogene mRNAs. Estrogen treatment resulted in rapid elevation of both EGF and EGFr mRNA levels, both of which were suppressed after tamoxifen treatment. Tamoxifen may exert its antiestrogenic effects by inhibiting EGF and EGFr, and myc protooncogene activity on the one hand, and by overexpression of the TGFB isotypes and their receptors on the other hand. With the proliferation cycle short circuited, tamoxifen-treated cells hypertrophied and differentiated to terminal cells by 24-48 h. Working hypotheses for the mechanisms of action of estrogen and tamoxifen are presented based on our data.

Human erythrocytes have binding sites for beta-endorphin.

Monoiodinated human beta-endorphin was found to bind specifically to human erythrocytes. Unlabeled beta-endorphin and beta-endorphin inhibited binding, but (-)naloxone, [D-Ala2, D-Leu5]-enkephalin, and leu- and met-enkephalin did not. Immunoelectron microscopy, using rabbit anti-beta-endorphin antibody, an antirabbit IgG secondary antibody, and complexed horseradish peroxidase, revealed that at low concentrations beta-endorphin binds to the cell surface. Electron spin resonance spectroscopy showed no effect of beta-endorphin on membrane fluidity. This receptor does not appear to conform to the characteristics of an opiate receptor.

Rat erythrocyte insulin receptors: radioreceptor assay and characterization.

Highly specific insulin receptors have been identified on the rat erythrocyte. A radioreceptor assay for the evaluation of these receptors has been developed, and the characteristics of these receptors have been investigated. Insulin receptor binding on the rat erythrocytes was found to be dependent on pH, temperature, time, and ionic strength. When incubated for 3½ hours at 15° C, 5.0 × 10(9) erythrocytes/mL from each of 10 rats were found to bind specifically 7.54 percent (±0.15 SEM) of 40 pg of (125)I-insulin. Specific binding was found to be a function of cell concentration. The pH optima for insulin binding were found to be 7.4 and 7.0 in the absence of cations. The presence of cations not only shifted pH optimum to 7.4 from 7.0, but also increased specific insulin binding.These observations suggest the stabilization of negatively charged groups on ligand and receptor, as well as providing a suitable ionic environment for the hormone-receptor interaction. Based on the resistance of rat erythrocytes to the pH of the external buffer, a simple method for determining the internal pH of rat red- blood cells is described. Scatchard analyses of insulin-binding data yielded curvilinear plots, and the number of receptor sites per cell was found to be 762 (±12.1 SD), as opposed to the large variation (410 ± 260 SD) in normal humans. The rat erythrocytes may serve as a useful, precise, sensitive, and efficient model system for future erythrocytic-receptor studies that would be difficult to obtain from human subjects.

Complementary DNA copies of leukemia and sarcoma virus RNA contain sequences of deoxycytidylate and deoxyguanylate.

Single-stranded complementary DNA (cDNA) of the RNA of Gazdar murine sarcoma virus, Gz-MSV/MuLV; Moloney murine leukemia virus, M-MuLV; mouse mammary tumor virus, MMTV; and simian sarcoma virus, SSV-1, were synthesized in endogenous reverse transcriptase reaction. Gz-MSV/MuLV cDNA was also synthesized in exogenous in vitro reverse transcriptase reactions. In the endogenous reaction, 30-35S, or 6.0- to 7.9-kilobase-length cDNA transcripts were synthesized in high yield. In comparison, transcripts synthesized in exogenous reactions were 6.7S, or 0.39 kilobases. The complementarity of the transcripts was verified by both RNA/DNA hybridization and protection studies. dG and dC sequences were detected in 50-77% of the cDNA molecules by affinity chromatography, by annealing and masking studies, and by resistance to S1 nuclease. dT and dA sequences were not detected in the transcripts. These findings are discussed in relation to the possible selective blocking of transcription of retrovirus genes without interfering significantly with the transcription of cellular genes.

Poly(U)-agarose affinity chromatography: specific, sensitivity selectivity, and affinity of binding.

These studies were done to determine four basic intrinsic properties of poly(U)-agarose affinity columns. Specificity of binding studies demonstrated that binding to these columns is highly specific with greater than 90% complementary binding and less than or equal to 3% noncomplementary binding. Sensitivity of binding studies indicated that a minimum sequence of 10 adenylates is required for detectable complementary binding. Selectivity of binding studies revealed that nonsequential adenylates in native RNAs and randomly distributed adenylates in synthetic poly(A)-poly(C) co-polymers did not bind to poly(U)-agarose affinity columns. Whereas, affinity of binding studies demonstrated that A=U complementary base pairing is independent of chain-lengths of greater than or equal to 25 adenylates and dependent of chain-lengths of less than 25 adenylates. Thus the data demonstrates that poly(U)-agarose affinity chromatography is scientifically sound and expedient for the detection and isolation of poly(A)-containing cellular and viral RNAs.

Erythrocytes: a new cell type for the evaluation of insulin receptor defects in diabetic humans.

Human erythrocytes have specific insulin receptors. When studied in an insulin radioreceptor assay, erythrocytes from adult-onset, nonobese diabetic subjects bound at least 42 percent less insulin than the normal subjects at insulin concentrations from 0.1 to 100 nanograms per milliliter. The diabetic subjects had 190 insulin receptor sites per cell as compared with the 380 insulin receptor sites per cell for the normal subjects. The deficit of insulin binding in the diabetic subject was thus associated with a fewer number of insulin binding sites per cell with little or no change in affinity. The erythrocyte is a readily available cell for the evaluation of cellular insulin receptor activity.

Streptococcus pyogenes preparation OK-432: immunoprophylactic and immunotherapeutic effects on the incidence of spontaneous leukemia in AKR mice.

An inactivated and lyophilized preparation of a low virulence strain (Su) of Streptococcus pyogenes (group A) was designated OK-432. When 2- and 5-month-old AKR mice were inoculated im with OK-432 twice weekly throughout their life-spans, spontaneous leukemias occurred later and at a lower incidence than in control groups. By virus neutralization and cytotoxicity tests and by immunoelectron microscopy, antibodies against virus and cell-surface antigens of transplanted AKR leukemia were not detectable in sera of nonleukemic mice of any group. Whereas sera from mice treated with OK-432 were the only positive for interferon, viremia was clearly demonstrated in control groups by reverse transcriptase assays of the plasma.

Enhancement of host immune response to cell surface antigens by a preparation of Streptococcus hemolyticus.

A preparation (OK-432) of a low virulence strain, Su, of Streptococcus hemolyticus (Group A) has been found to possess 2 different effects on tumor cells: a) A direct pharmacological effect like other antitumor drugs, and b) an indirect effect through the enhancement of the host immune response. In order to examine the second effect, 2 different antigen systems were used, Gross (G) cell surface antigens (GCSAs) and the cell surface differentiation antigen PCl. At certain ages, untreated C57BL/6 and BALB/c mice occasionally produce natural antibodies to GCSAs and PCl, respectively. When the mice were injected intraperitoneally (i.p.) with OK-432 at 2 months of age, they produced natural antibodies to GCSAs or to PCl antigen, unlike untreated mice of the same strains at the same ages (negative controls), which produced no antibodies. The specificity of the antibodies was carefully examined by cytotoxicity tests. In addition, the activity of the natural antibodies was not neutralized with OK-432 itself. We have proven that OK-432 enhances the production of natural antibodies to specific cell-surface antigens.

Polyriboadenylate sequences at the 3'-termini of ribonucleic acid obtained from mammalian leukemia and sarcoma viruses.

The location of poly(A) sequences in the RNA of mammalian RNA-tumor viruses was determined by enzymatic analyses. The 56-64S viral genomic RNAs, the 20-40S viral subunit RNAs, and the 4-5S poly(A) sequences excised from these viral RNAs were subjected to either hydrolysis with a 3'-OH specific exoribonuclease from Ehrlich ascites tumor cells or phosphorolysis from the 3'-termini with polynucleotide phosphorylase from Micrococcus luteus. Purified adenosine-labeled poly(A) fragments, excised from genomic viral RNAs by RNase A and T(1) digestion, were hydrolyzed with the 3'-OH specific exoribonuclease for various periods of time. Poly(U) filter binding studies of the residual poly(A) indicated that 97% of the poly(A) fragments were hydrolyzed. Adenosine-labeled genomic and subunit viral RNAs and excised poly(A) fragments were phosphorolyzed from their 3'-termini for various periods of time with polynucleotide phosphorylase. The degree of phosphorolysis was monitored by poly(U) filter binding studies, and CCl(3)COOH insolubility and solubility determinations. There was an initial preferential rate of phosphorolysis of the poly(A) sequences of genomic and subunit viral RNAs as compared to the total adenosine-labeled viral RNAs. The data from these two different enzymatic mechanisms of action indicated conclusively that the poly(A) sequences were located at the 3'-termini of genomic and subunit viral RNAs.

Detection of naturally occurring antibodies to RNA-dependent DNA polymerase of murine leukemia virus in kidney eluates of AKR mice.

Specific antibodies to the RNA-dependent DNA polymerase (reverse transcriptase) of murine type C viruses have been isolated from the renal glomeruli of both leukemic and nonleukemic AKR mice where they presumably had been deposited as immune complexes. The antibodies were shown to have sedimentation coefficients of 26S to 28S and 5S to 7S on sucrose rate zonal centrifugation. Inactivation with monospecific antisera to various mouse immunoglobulins identified antibodies as being in both immunoglobulin (IGM) and IgG classes. In addition, these antibodies only reacted with the reverse transcriptase from murine and feline type C viruses, but not the polymerase from avian myeloblastosis virus (AMV). Our results provide additional evidence for the lack of immunological tolerance and demonstrate the presence of another immune complex system in AKR kidneys.

Characterization of RNA from noninfectious virions produced by sarcoma positive-leukemia negative transformed 3T3 cells.

RNA from noninfectious virions produced by two established clonal lines of sarcoma positive-leukemia negative (S+L-)-transformed 3T3 cells has been characterized. RNA from virions or nucleoids of S+L--(C243) cells consisted of three to four sizes: +/-44 S (6%), 28 S (17%), 18 S (38%), and <18 S (39%). 28S virion RNA contained some virus-specific information demonstrable by RNA.DNA hybridization with a DNA probe derived from the RNA-directed DNA polymerase product of murine sarcoma-leukemia virus, while parallel studies indicated that 28S ribosomal RNA from ribosomal subunits of transformed and nontransformed 3T3 cells did not contain virus-specific information. In contrast to the S+L-(C243) virions, RNA from virions or nucleoids of S+L-(D56) cells consisted of five sizes: 80 S (6%), 68 S (8%), 56 S (5%), 28 S (28%), and <28 S (53%). Thermal dissociation studies suggested that this S+L- genome is comprised of 28S RNA subunits. From these studies we postulate that the 28S viral RNA is most probably the monomeric genome of S+L- virions.