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Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping.

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) persists despite a vigorous virus-specific host immune response, and causes adult T-cell leukemia and lymphoma in approximately 2% of infected individuals. Here we report that HTLV-1 has evolved a genetic function to restrict its own replication by a novel post-transcriptional mechanism. The HTLV-1-encoded p30(II) is a nuclear-resident protein that binds to, and retains in the nucleus, the doubly spliced mRNA encoding the Tax and Rex proteins. Because Tex and Rex are positive regulators of viral gene expression, their inhibition by p30(II) reduces virion production. p30(II) inhibits virus expression by reducing Tax and Rex protein expression.

The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex mRNA compared to pX-tax-orfII and pX-rex-orfI mRNAs, but did not affect exon skipping. Ectopic expression of hnRNP A1 did not affect pX splice site utilization, but increased exon skipping, as the level of pX-p21rex mRNA was increased by almost 10-fold.

Human T-cell leukemia virus type 1 (HTLV-1) infection profoundly alters T-cell gene expression, and the dysregulated synthesis of cytokines could influence the course and pathologic consequences of infection. In the process of screening T-cell lines for T helper 1 (Th1) and Th2 cytokine mRNAs, we observed that interleukin-13 (IL-13) mRNA was highly expressed in HTLV-1-infected, IL-2-dependent T-cell lines. IL-9 and interferon gamma (IFN-gamma) mRNAs were also expressed at high levels in chronically infected cell lines. IL-5 mRNA was detected in 60% of the HTLV-1-infected cell lines, but mRNAs for IL-4, IL-10, IL-2, and IL-15 were either below detection limits or did not correlate with HTLV-1 infection. Transcriptional activation of the IL-13 promoter by the HTLV-1 Tax trans-regulatory protein was demonstrated in Jurkat T cells transiently transfected with an IL-13 promoter-reporter plasmid. The clinical relevance of these observations was demonstrated by immunofluorescent staining and flow cytometry of lymphocytes obtained from HTLV-1-infected patients. These studies revealed that IL-13 production was directly related to the level of Tax expression in the infected CD4+ T cells soon after in vitro culture. As IL-13 plays key roles in tumor immunosurveillance, asthma, and central nervous system inflammation, it may contribute to the pathophysiology of HTLV-1-associated diseases.

In order to analyze biochemical properties of Xenopus bone morphogenetic protein-1 (XBMP-1), rabbit antiserum (alpha-B1) was raised against a synthetic peptide (P1) corresponding to a hydrophilic N-terminal region. XBMP-1B (Xtld) synthesized in the reticulocyte lysate was successfully immunoprecipitated by this antiserum. This precipitation was completely blocked when P1 was added to the reaction, indicating that alpha-B1 recognized XBMP-1B specifically. In Western blot analysis, two distinct sizes of protein (107 and 34 kD) were detected in hind limbs in metamorphosing animals. Both proteins were detected in various adult tissues such as lung, liver, kidney, heart, muscle, intestine, brain, and testis. The mixing of the liver and muscle extracts, and the following detection of immunoreactive proteins suggested that the 34 kD band was a proteolytic product of the 107 kD protein. In the embryonic extracts from the unfertilized egg (stage 0) to swimming tadpoles (stage 40), a 63 kD protein was detected in addition to the 107 kD protein. We also showed that the 107 kD protein was much more expressed in the animal half of the unfertilized eggs than in the vegetal half, but that it was ubiquitously expressed in the gastrula embryos. We suggest that the 63 and 107 kD proteins correspond to full-length proteins encoded by XBMP-1A and XBMP-1B genes, and these proteins are expressed in embryo and in various adult tissues.

Microsomal triglyceride transfer protein (MTP) plays a central role in the assembly and secretion of apoB-containing lipoproteins. In this study, we investigated the effect of ethanol on the expression of the large subunit of MTP in a human liver hepatoma cell line, the HepG2 cells. Exposure of HepG2 cells to low concentrations of ethanol reduced MTP mRNA levels in a concentration- and time-dependent manner. The level of MTP mRNA decreased significantly (P<0.05, -26% relative to pretreatment control) when the concentration of ethanol in the culture medium was 50 ppm (0.005%, v/v). Maximal suppression (-50%) was observed at 100 ppm ethanol; the MTP mRNA levels remained at 50% of control when the ethanol concentration was raised to 10,000 ppm. Furthermore, a 10-day ethanol treatment caused a significant 50% decrease in the MTP activity and apoB secretion rate in HepG2 cells. To investigate the molecular mechanisms underlying this phenomenon, we examined the effect of ethanol on the promoter activity of the MTP gene. Transient transfection analysis of human MTP promoter-driven luciferase gene expression showed that ethanol down-regulates MTP promoter activity in a manner parallel to that observed for mRNA levels. Deletion analysis suggested that the MTP promoter sequence contains a negative ethanol response element -612 to -142 bp upstream of the transcription start site. To evaluate the in vivo relevance of the effect of ethanol on MTP mRNA levels, rats were given a single oral dose of ethanol, with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats receiving 1 or 3 g/kg of ethanol exhibited substantially lower hepatic and intestinal MTP mRNA levels. Taken together, these results strongly suggest that ethanol can modulate the secretion of apoB-containing lipoproteins by down-regulating the expression of MTP large subunit, primarily through inhibiting the transcription of the MTP gene.

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-alpha or IL-6 mAb. We asked, therefore, whether IFN-alpha-induced growth inhibition involved IL-6. IFN-alpha and mAb against IL-6, the IL-6R alpha-(gp80) or beta-chain (gp130) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-alpha-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-alpha drastically curtailed expression of the IL-6R alpha- and beta-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-alpha, but not IL-6, greatly decreased gp80 and, to a lesser extent, gp130 mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-alpha-induced IL-6 predominantly down-regulated membrane-bound gp130. IFN-alpha-mediated decrease of gp80 levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-alpha inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R.

The interaction between human interferon (IFN)-alpha or IFN-beta with its receptor was originally described as the binding to a single class of high-affinity receptors. However, more recently, biphasic Scatchard plots as well as multiple IFN-alpha receptor cross-linked complexes have been reported. In this study using the Daudi B lymphoblastoid cell line, two primary IFN-alpha receptor cross-linked complexes with apparent Mr of 115 and 135 kilodaltons (kDa) were obtained. Both complexes were observed under a variety of cross-linking conditions, including the addition of a mixture of protease inhibitors throughout the binding reaction and solubilization of the cells. These two complexes appear to be caused by the binding and cross-linking of 125I-rIFN-alpha A to two separate proteins because we also observed two IFN-alpha binding proteins using a ligand-blotting technique. At low concentrations of 125I-rIFN-alpha A, it was found that the intensity of the signal in the 135-kDa cross-linked complex was greater than that of the 115-kDa complex. Addition of increasing concentrations of unlabeled rIFN-alpha A to a 4 degrees C binding reaction reversed the ratio in intensities of the two complexes. Moreover, after pretreatment of the cells at 37 degrees C with low concentrations of unlabeled rIFN-alpha A, there was preferential down-regulation of both the 135-kDa complex and the higher affinity binding component of the biphasic Scatchard plot. These results suggest that the 135-kDa complex represents the binding of 125I-rIFN-alpha A to a protein having higher affinity for IFN than the protein that gives rise to the 115-kDa complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The hybrid recombinant human interferon (IFN) rIFN-alpha A/D was radioiodinated. Specific binding of [125I]rIFN-alpha A/D was observed with both human and murine cell lines. The binding of [125I]rIFN-alpha A/D to human Daudi cells had similar characteristics to the previously described binding of [125I]rIFN-alpha A or -alpha 2. The following lines of evidence demonstrated that [125I]rIFN-alpha A/D bound with high affinity to the same receptor on murine cells as murine IFN-alpha and -beta: (i) the binding of [125I]rIFN-alpha A/D to murine LBRM cells was inhibited to a similar extent by natural murine IFN-alpha, natural murine IFN-beta, and rIFN-A/D; (ii) the Kd (approximately 2 X 10(-10) M) obtained from both competition experiments and saturation binding experiments with [125I]rIFN-alpha A/D was comparable to the previously reported Kd for the binding of natural murine IFN-alpha and -beta to other murine cell lines; (iii) the size of the cross-linked [125I]rIFN-alpha A/D receptor complex formed on murine LBRM cells was similar to the previously reported cross-linked complex formed after binding radioiodinated natural murine IFN-beta to other murine cell lines. Due to the current lack of readily available recombinant murine IFN-alpha or -beta for radiolabeling and the previously demonstrated biological activity of rIFN-alpha A/D on murine cells, [125I]rIFN-alpha A/D should prove to be a useful reagent for further studies of murine IFN receptors.

Combination treatment of SKCO1 human colon carcinoma cells with beta ser-interferon (IFN-beta ser) and gamma-interferon (IFN-gamma) results in a synergistic antiproliferative effect. The role of IFN-beta ser and IFN-gamma receptor modulation was investigated as a possible mechanism for this response. IFN-gamma (0.05-50 ng/ml) pretreatment of SKCO1 cells for 24 h decreased specific binding of 125I-IFN-beta ser by 35-60%. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-gamma showed that this reduction in binding was due to a decreased receptor affinity (control cells, Kd = 46 +/- 1.6 pM; IFN-gamma-treated cells, Kd = 106 +/- 6 pM, n = 2) rather than a significant change in receptor number (receptor number/control cell = 1214 +/- 471, receptor number/IFN-gamma treated cell = 1118 +/- 153, n = 2). In contrast, pretreatment of SKCO1 cells with IFN-beta ser (5 ng/ml) resulted in slight (10-35%) increases in 125I-IFN-gamma-specific binding. Scatchard analysis of binding data obtained following 24-h treatment with 5 ng/ml IFN-beta ser showed a decrease in binding affinity (control cells, Kd = 28 +/- 7 pM; IFN-beta ser-treated cells, Kd = 38 +/- 7 pM, n = 2) and a 32% increase in IFN-gamma receptor sites (receptor number/control cell = 4257 +/- 464, receptor number/IFN-beta ser-treated cell = 5570 +/- 730; n = 2). 125I-IFN-gamma internalization studies performed at 37 degrees C confirmed the cell surface binding assays; IFN-beta ser-treated cells internalized 30-50% more labeled IFN-gamma than untreated cells. However, it is unlikely that differences in binding and internalization of this magnitude play a primary role in the synergistic antiproliferative effect of IFN-gamma with IFN-beta ser in SKCO1 cells. Biochemical modulation at sites distal to the ligand receptor interaction should be investigated.

In vitro synthesis of alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) utilizing 14C-L-leucine and 14C-L-lysine incorporation in fresh testicular cancer tissue of five patients was demonstrated. Radioimmunoassay, immunoprecipitation of 14C-HCG and 14C-AFP, and quantitation of AFP and HCG. This in vitro synthesis of AFP and HCG was correlated with immunocytochemical staining in the cancer cells of the five patients.

A human lung tumor-associated protein has been purified from an extract of a human small cell carcinoma of the lung and shown by Ouchterlony double diffusion analysis to be antigenically identical to a component which was previously demonstrated in 84 of 98 lung tumor extracts of all histological types but absent from extracts of normal adult and fetal lung, other normal tissues, and tumors of other organs. These studies utilized xenoantisera raised against a pool of lung tumor extracts which were exhaustively adsorbed with normal serum and tissue extracts. A radial immunodiffusion assay developed for the antigen permitted its quantitation throughout the course of isolation. Purification was accomplished by ion-exchange chromatography, gel filtration, and affinity immunoadsorption. By ion-exchange chromatography, the proteins appeared to be quite heterogeneous, with immunological reactivity detected in three different peaks. However, all the active components were immunologically identical. Gel filtration of the major antigenic component from diethylaminoethyl cellulose similarly demonstrated a further fractionation into several active, immunologically identical forms. These results suggest a charge-size isomeric relationship among the various forms, all of which possess a common and identical antigenic site. The major component was isolated throughout the purification scheme. The final product represented 9% of the input activity, produced a single, although broad, protein-staining region on 7% polyacrylamide gels which was coincident with antigenic activity, and exhibited immunological identity with the antigen in the crude extract as well as with that in an extract from another lung tumor.

Five monkeys were treated ip with N-nitrosodiethylamine (DENA), and one was treated with 1-nitrosopiperidine (PIP), starting within 2 months of birth, until hepatocellular carcinoma (HCC) developed. All animals except the PIP-treated monkey had much elevated serum alpha-fetoprotein (AFP) values. Fresh, minced, biopsy-derived tumor was cultured with L-[14C]leucine and L-[14C]lysine. Synthesis of AFP was determined by radioimmunoassay and by specifically precipitable [14C]AFP. Good agreement between these two parameters was obtained for the 4 DENA-induced tumors synthesizing AFP in culture. Tumor from 1 DENA-treated monkey did not synthesize AFP. In addition, neither normal liver nor tumor from the PIP-treated monkey showed AFP synthesis. Rates of synthesis were 0.37-5.50 ng AFP/mg tumor/day, or 0.0012-0.0183 pg AFP/cell/day (if one assumes 3.0 X 10(5) cells/mg tissue) over 48 or 72 hours. Different nodules from the same animal had similar rates of synthesis. For tumors that synthesized AFP in culture, a positive correlation was generally found between rate of synthesis and serum AFP level. The rate of in vitro AFP synthesis observed was lower than that of immunoglobulin synthesis in human myeloma or of AFP synthesis in a rat HCC, but it was close to the estimated rate of AFP synthesis in a monkey HCC line in long-term culture.

A solid-phase, enzyme-linked immunosorbent assay (ELISA) for a human lung tumor-associated antigen (LTA) was based on immobilized LTA that was detected with the use of an antiserum raised in a goat against a highly purified antigen preparation. Bound goat antibodies were detected in a series of steps that included incubation with a) biotinylated rabbit antibodies to goat immunoglobulins, b) glucose oxidase conjugated to avidin, and c) peroxidase and the substrates glucose and 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid). The absorbance of the final product was measured at 405 nm, and its formation was dependent on substrate incubation time and antibody concentration. The antigen was immobilized and highly purified, and the goat antiserum was bound to and eluted from an immobilized crude antigen column before use. The ELISA could detect less than 1 ng antigen and was able to discriminate extracts of normal lung tissue from those of lung tumor. As was found earlier with a radioimmunoassay for the same antigen, normal human serum could inhibit in the ELISA but only when used at high concentration, indicating levels of antigen or antigen-like activity in the 100-200 ng/ml range. With the use of this assay, 3 lung cancer patients were monitored 6-12 months prior to death. In all 3 patients, LTA levels rose dramatically 2-4 months before the patients died; in 2 patients the levels exceeded 3,000 ng/ml just before death. In contrast, in 2 of these patients, carcinoembryonic antigen levels remained essentially unchanged, with no more than a twofold increase prior to death.

A human lung tumor-associated antigen (LTA), previously isolated from a small cell carcinoma, was further studied after labeling with N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate. Two immunoreactive forms of the labeled antigen were observed and isolated after electrophoresis in 7% polyacrylamide gels. These components, referred to as LTA-I and LTA-II in order of mobility, were judged homogeneous by gel electrophoresis, G-200 gel filtration, size exclusion high-performance liquid chromatography, and sedimentation velocity analysis. The latter three techniques produced identical profiles for both forms of the LTA. Sephadex chromatography and high-performance liquid chromatography analyses indicated the mass of the antigens to be approximately 140,000 to 150,000 daltons with a D20,w of 4.2 to 4.3 x 10(-7) sq cm/sec. The S20,w values for both were 4.5 to 4.6S. Sodium dodecyl sulfate gel electrophoresis of LTA-II gave a single component with a molecular weight of 81,700, while LTA-I had a major component identical in size to LTA-II and two minor components with molecular weights of 50,000 and 27,700, respectively. The isoelectric point of LTA-II (peaks at pH 2.6 and 3.2) generally was more acidic than LTA-I (major component centered at pH 4.7, minor component centered at pH 3.1). A radioimmunoassay, with a useful detection range of from 1 to 100 ng/ml, was developed with LTA-I. This assay was used to determine the concentration of LTA in the sera of normal and lung cancer patients. Fifteen normal sera had a mean of 17 +/- 22 (S.D.) ng/ml, with none greater than 83 ng/ml (+3 S.D.). Thirteen lung cancer patients with Stage I (i.e., localized disease) had a mean of 187 +/- 219 ng/ml, with the means for 7 of 13 patients being greater than 83 ng/ml; 15 lung cancer patients with Stage III, more extensive disease, had a mean of 277 +/- 252 ng/ml, with the means for 12 of 15 patients being greater than 83 ng/ml. This antigen may be useful for the early detection or monitoring of lung cancer.

After binding to specific cell surface receptors, interferon-alpha (IFN-alpha) along with its receptor is internalized by the cells. However, the physiological significance of the internalization of IFN is not known. We have found that the lectin concanavalin A (ConA), which does not inhibit the binding of 125I-rIFN-alpha A, inhibits both the internalization of 125I-rIFN-alpha A and the rIFN-alpha A-induced increase in the levels of 2',5'-oligo(A) synthetase mRNA and enzymatic activity in the B lymphoblastoid cell line Daudi. The reduced level of IFN-induced 2',5'-oligo(A) synthetase in ConA-treated cells was due neither to direct inhibition of the enzymatic activity nor to generalized inhibition of protein or RNA synthesis. The dose-response curves were similar for the effect of ConA to inhibit 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction. The correlation between the ConA-mediated inhibition of both 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction suggests that internalization of rIFN-alpha A plays a role in the responses to rIFN-alpha A. However, since ConA inhibits protein mobility in the plasma membrane, it is possible that ConA is also preventing aggregation of IFN receptors or interactions between IFN receptors and signal transducing proteins in the plasma membrane that may be necessary for responses to IFN.

The expression of interferon (IFN) receptors was studied on freshly isolated human lymphocytes from normal donors. Highly enriched populations of small resting T lymphocytes and large granular lymphocytes (LGL) were found to constitutively express high-affinity receptors for IFN-alpha and IFN-gamma. Both types of lymphocytes also had lower-affinity IFN-alpha binding sites. T lymphocytes had a mean of 230 IFN-alpha and 520 IFN-gamma high-affinity receptors per cell, whereas LGL had 520 IFN-alpha and 760 IFN-gamma receptors. However, because LGL were larger than the T lymphocytes, the IFN receptor density was similar on the two types of lymphocytes. The affinity of binding was similar on the two types of normal lymphocytes and on the cultured lymphoblastoid cell line Daudi. The number of IFN receptors per cell and the affinities of the IFN-receptor interactions varied little among the normal donors. Both the freshly isolated normal lymphocytes and the cultured cell line Daudi had separate receptors for type I (alpha and beta) and type II (gamma) IFN. Taken together, our data indicate that two types of freshly isolated normal lymphocytes constitutively express IFN receptors that are similar to those present on the lymphoblastoid cell line Daudi derived from a patient with Burkitt's lymphoma.

The human type I interferon (IFN) receptor was characterized by ligand blotting. In this method, plasmalemma proteins or detergent-lysed whole-cell extracts from human Burkitt lymphoma cell lines were separated on polyacrylamide gels and subsequently transferred onto nitrocellulose sheets. Probing the blots with 3 x 10(-10) M 125I-labeled recombinant IFN-alpha A (125I-rIFN-alpha A) revealed an IFN-alpha-binding protein with an apparent molecular mass of 95 kDa (p95). Performing the electrophoretic run under reducing conditions completely abrogated the signal on the blot, indicating that the type I IFN receptor contains a disulfide bond essential for IFN binding. Optimal binding of 125I-rIFN-alpha A occurred at pH 9. The specificity of the binding reaction was established by simultaneously adding an excess of unlabeled IFN during incubation of the blots with 125I-rIFN-alpha A. The addition of either unlabeled IFN-alpha or IFN-beta, but not IFN-gamma, abolished the binding of 125I-rIFN-alpha A to p95. 125I-IFN-gamma at 1.25 x 10(-11) M bound to two proteins distinct from p95 with apparent molecular mass of 92 and 87 kDa, respectively. Saturability of 125I-rIFN-alpha A binding was demonstrated by probing a constant amount of membrane proteins with increasing amounts of 125I-rIFN-alpha A. Scatchard analysis of the binding data yielded an apparent Kd of 5.4 x 10(-10) M for the immobilized type I IFN receptor. The expression of p95 on IFN-alpha-resistant and -sensitive cells was indistinguishable. We conclude that p95 is the IFN-alpha/beta receptor and that two proteins (p92 and p87) can specifically bind IFN-gamma. These results indicate that ligand blotting is a versatile method for characterization of unmodified IFN receptors and IFN-receptor interaction and could also provide a new investigational approach for other cytokine receptor systems.

A human lung tumor-associated antigen was purified from a saline extract of a lung adenocarcinoma. The antigen was demonstrated in extracts of lung adenocarcinoma. The antigen was demonstrated in extracts of lung tumors with the use of an absorbed antiserum by double-diffusion immunoprecipitation. The antiserum did not react with extracts of normal lung or other normal tissues, and the antigen was immunologically distinct from other tumor-associated antigens. Purification was achieved by antibody affinity chromatography and preparative polyacrylamide gel electrophoresis. Isolation procedures were monitored by immunoreactivity with absorbed monospecific antiserum. The antigen was labeled with 125I and judged homogeneous by 1) polyacrylamide gel elecrophoresis in detergent and nondetergent gels, 2) molecular sieve chromatography, 3) ion exchange chromatography, and 4) sucrose gradient sedimentation analysis. A molecular weight of 77,000 was calculated from the s20.w value of 4.24S and from the D20.w value of 5.0X10(-7) cm2/sec. Sodium dodecyl sulfate gel electrophoresis indicated a subunit molecular weight of 42,000. The Stokes radius of the antigen was 40 A and the frictional ratio was 1.42, indicating a nonspherical molecule. The purified radioiodinated antigen could be quantitatively precipitated with specific antiserum.

Freshly isolated normal human T lymphocytes constitutively expressed receptors for interferon-alpha (IFN-alpha) and IFN-gamma. Upon activation, the number of IFN-alpha receptors increased, paralleling the increases in cell size to give a nearly constant density of IFN-alpha receptors. In contrast, activation of T lymphocytes with phytohemagglutinin (PHA), concanavalin A (ConA), or phorbol myristate acetate (PMA) for 18 h decreased the specific binding of radiolabeled [Cys-Tyr-Cys] rIFN-gamma to acid-washed cells. Scatchard analysis demonstrated that the decreased binding was due to a reduction in the number of high affinity IFN-gamma receptors. Moreover, resting T lymphocytes had a single class of high-affinity IFN-gamma receptors, whereas the activated cells appeared to have both high- and lower-affinity IFN-gamma binding sites. When normalized for differences in cell size, the number of high-affinity IFN-gamma receptors per unit cell surface area was approximately twofold lower on activated than on resting T lymphocytes. At least part of the decrease was due to receptor downregulation by endogenously produced IFN-gamma, since monoclonal antibodies to IFN-gamma prevented a large portion of the PHA-induced decrease in IFN-gamma receptors.

The pregnant rhesus monkey's (Macaca mulatta) potential as a model for understanding the dynamics of alpha-fetoprotein (AFP) metabolism in human pregnancy was evaluated. AFP levels in maternal and fetal serum and amniotic fluid were determined by radioimmunoassay. Significant correlations were found between decreasing maternal serum, fetal serum and amniotic fluid AFP concentrations and increasing gestational age. However, these data are not consistent with the AFP changes reported in human pregnancy. It appears that this animal has limited applicability as a model in this aspect of human pregnancy.

A human lung tumor-associated antigen was purified to homogeneity from a crude cell-free extract of a human lung adenocarcinoma using standard biochemical procedures. In order to facilitate monitoring the recovery of antigen, trace amounts of previously purified and radioiodinated antigen from another lung tumor were added to the crude extract. The purified antigen was a glycoprotein and contained sialic acid. The antigen had a molecular weight of 76,000 and appeared to contain three subunits, each with a molecular weight of 25,000. The antigen had the following physical properties: Stokes radius, 39.4 A; S20,w, 4.24 S; D20,w, 5.15 x 10(-7) cm2 S-1; and a frictional ratio of 1.40. In addition, the purified, radioiodinated antigen retained complete immune reactivity since it could be quantitatively precipitated with specific immune serum. All of these properties were in close agreement with the properties of another antigen which was purified from a separate human lung tumor. Thus, it appeared from the biochemical and immunochemical criteria presented in this report that a common and identical antigen was isolated from two distinct human lung tumor extracts.

A human lung tumor-associated antigen, previously purified to apparent homogeneity from an extract of a small cell tumor, was radioiodinated with Bolton-Hunter reagent for use in a competitive protein-binding radioimmunoassay. A panel of 215 sera was assembled from normal individuals and pretreatment patients with lung cancer, benign lung disease, and nonlung cancers, and lung tumor antigen in each was quantitated using the radioimmunoassay. The mean of normals was 0.92 +/- 0.43 (S.D.) microgram/ml (n = 88), and values greater than 2 standard deviations above the mean (1.78 micrograms/ml) were considered positive. Positive rates in lung cancers of the following histological types were found: adenocarcinoma, 60% (9 of 15); squamous cell, 42% (13 of 31); large cell, 17% (3 of 18); and small cell, 19% (3 of 16). In addition, 13% (3 of 23) of other cancers, 0% (0 of 24) of benign lung disease, and 2% (2 of 88) of normals were positive. Approximately one-third of Stage 1 patients in the squamous cell and adenocarcinoma groups were positive while two-thirds of patients with more advanced Stage III disease in these categories showed elevations.

A human lung tumor-associated antigen has been identified by Ouchterlony analysis in 86% of lung tumor extracts of all histologic types. It was not detected by this method in extracts of normal adult or fetal lung, 12/13 other tumors, normal tissues or in normal serum. The antigen was purified from the extract of an undifferentiated small cell lung carcinoma by DEAE-cellulose. Sephadex G-200, and antibody affinity chromatography. The purified antigen exhibited size and charge heterogeneity, yet all isolated forms produced precipitin lines of identity with each other, as well as with an extract of an unrelated squamous cell carcinoma of the lung. The major form had a Mr of 150 000 by gel filtration and 81 700 by detergent gel electrophoresis. By radioimmunoassay, elevated levels of antigen were detected in 0/15 normal sera, 7/13 stage I and 12/15 stage III lung cancer sera. The urine of a different patient with a small cell lung carcinoma contained a related antigenic activity, but the major component was 30 000-50 000 daltons. The antigen has also been detected in extracts of a continuous cell line from a large cell carcinoma of the lung (ChaGo). The antigen is being evaluated for its usefulness as a lung tumor marker.

In order to examine the relative usefulness of measurements of oncoplacental proteins as tumor markers in patients with nonseminomatous germ cell tumors, the authors measured alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), pregnancy-specific beta 1-glycoprotein (SP1), human placental lactogen (hPL), and placental cystine aminopeptidase (oxytocinase, CAP) in serial blood samples obtained from 26 men with these neoplasms. HCG and AFP were each elevated in 62% of the patients and both were elevated in 38%. SP1 and hPL were increased in 31% and 12%, respectively. None of the patients had elevated CAP activity. Serum hCG and SP1 concentrations were strongly correlated (r = 0.78, P less than 0.001). No patient had an elevated SP1 without a concomitant elevation in serum hCG. Serial measurements of hCG and SP1 indicated that they were concordant in five of the eight patients in whom both were elevated, and AFP and hCG were concordant in only one half of the ten patients in whom both markers were elevated. The number of patients with hPL elevations were too few for meaningful comparison of this marker with the others. These results indicate that measurements of SP1, hPL, and CAP do not provide additional useful information over that obtained from measurements of hCG and AFP in patients with nonseminomatous germ cell tumors.

The prenatal diagnosis of anencephaly and spina bifida (neural tube defect, NTD) through amniotic fluid analysis for alpha-fetoprotein (AFP) is gradually gaining clinical recognition. AFP concentrations were determined in 237 amniotic fluids from normal pregnancies ranging between 7 and 42 weeks of gestation. A steady decline in AFP from 26 mug/ml at 7-9 weeks to 155 ng/ml at term is observed. AFP concentration was determined in 35 amniotic fluids from 33 confirmed neural tube defective pregnancies. In 14 cases where amniotic fluid was examined prior to the 26th week of gestation. AFP was markedly elevated when compared with the normal range of the same gestational period. In 21 amniotic fluids past the 26th week, 17 cases (85-) had markedly elevated AFP levels; however, 2 cases of anencephaly, 1 of spina bifida, and 1 of hydrocephaly gave levels within the normal range. It is concluded that elevated AFP in the amniotic fluid is a reliable but nonspecific marker for open neural tube defects prior to the 26th week of pregnancy, but may become normal after the 26th week in a small percentage of patients.

Treatment of Daudi B lymphoblastoid cells with interferon (IFN)-alpha or -beta has been reported (Yap, W. H., Teo, T. S., and Tan, Y. H. (1986) Science 234, 355-358) to cause a transient increase in the level of diacylglycerol, which is the endogenous activator of protein kinase C (PK-C). To assess the role for PK-C in the induction of 2-5A synthetase mRNA and activity by IFN-alpha in Daudi cells, we have examined the effects of PK-C inhibitors and activators. We have found that the PK-C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), strongly inhibits the induction of 2-5A synthetase mRNA and activity by recombinant IFN-alpha A (rIFN-alpha A) and that this inhibition appears to be at a post-transcriptional level. The inhibition by H7 could not be attributed to a generalized decrease in macromolecular synthesis or to inhibition of the binding or internalization of rIFN-alpha A. Moreover, pretreatment of Daudi cells for 24 h with phorbol esters to down-regulate or desensitize PK-C substantially inhibited the subsequent induction of 2-5A synthetase mRNA and activity by rIFN-alpha A. These data suggest that PK-C activity is required for the induction of 2-5A synthetase by rIFN-alpha A. However, phorbol esters, which are potent PK-C activators, did not induce 2-5A synthetase. Taken together, our data indicate that a functional PK-C is required for 2-5A synthetase induction by rIFN-alpha A at a post-transcriptional level in Daudi cells but that activation of PK-C is not sufficient for induction of this enzyme.

As a preliminary step in the identification and isolation of antibodies to human cancers, we have developed a sensitive and convenient assay for antibody binding to cellular antigens. The basis for the method is antibody binding to glutaraldehyde-fixed cells (AbGfC) and quantitation with radioiodinated staphylococcal protein A (SpA). Glutaraldehyde fixation of intact cells, which does not appear to effect the ability to form antigen-antibody complexes, provides a convenient and standard supply of target cells which may be stored at 4 degrees C and used in the assay over a period of several months. The amount of antibody specifically bound to the cells is quantitated by the addition of 125I-labeled SpA. The sensitivity of the method was compared with two complement-dependent cytotoxicity methods (trypan blue exclusion and 51Cr release assays) and tested with two antisera to human lung cancer and one antiserum to a membrane antigen of a murine lymphoma. These comparisons indicated much greater sensitivity when compared with the trypan blue exclusion assay and equivalent sensitivity with greater dose response characteristics when compared with the 51Cr release assay.

A human lung tumor-associated antigen (LTA) previously purified from a primary lung tumor has been identified in the sera of lung cancer patients. Frequencies of LTA elevations in lung cancer were: adenocarcinoma, 60%; squamous cell carcinoma, 42%; large cell carcinoma, 17%; and small cell carcinoma, 19%; normals, 2%; benign lung disease, 0%; and non-lung malignancies, 13%. The antigen was also shown to be produced by seven of the eight human lung tumor cell lines that were examined. A preliminary small-scale purification was attempted on an extract from one of these lines, ChaGo, which yielded a smaller and more basic form of LTA but which possessed similar, if not identical, antigenic activities as primary tumor LTA.

The Daudi B lymphoblastoid cell line was previously demonstrated to be highly sensitive to the antiproliferative effect of recombinant interferon-alpha A (rIFN-alpha A). In the present study, glucocorticoid hormones were shown to act synergistically with rIFN-alpha A to further increase the sensitivity of Daudi cells to rIFN-alpha A. At 10(-6) M, dexamethasone, prednisolone, or hydrocortisone alone had little effect on Daudi cell growth, but they greatly potentiated the antiproliferative activity of rIFN-alpha A. The synergy between rIFN-alpha A and glucocorticoids on Daudi cells was not related to the inhibitory effects of glucocorticoids on prostaglandin or leukotriene synthesis, since no synergy was observed between rIFN-alpha A and indomethacin or nordihydroguaiaretic acid. Glucocorticoids and rIFN-alpha A also had appreciable synergistic antiproliferative effects on two out of five other IFN-sensitive lymphoid cell lines. When Raji B lymphoblastoid cells, which were quite resistant to the antiproliferative effect of rIFN-alpha A, were treated with the combination of glucocorticoids and rIFN-alpha A, no significant synergistic effects were observed. The synergistic antiproliferative effects of glucocorticoids and rIFN-alpha A observed with some IFN-sensitive lymphoid cell lines in this in vitro study may have clinical relevance in the treatment of certain lymphoid malignancies that are sensitive to rIFN-alpha A therapy.