Chronic hepatitis C infection and liver disease in HIV-coinfected patients in Asia.

Data on markers of hepatitis C virus (HCV) disease in HIV-HCV-coinfected patients in resource-limited settings are scarce. We assessed HCV RNA, HCV genotype (GT), IL28B GT and liver fibrosis (FibroScan® ) in 480 HIV-infected patients with positive HCV antibody in four HIV treatment centres in South-East Asia. We enrolled 165 (34.4%) patients in Jakarta, 158 (32.9%) in Bangkok, 110 (22.9%) in Hanoi and 47 (9.8%) in Kuala Lumpur. Overall, 426 (88.8%) were male, the median (IQR) age was 38.1 (34.7-42.5) years, 365 (76.0%) reported HCV exposure through injecting drug use, and 453 (94.4%) were on combination antiretroviral therapy. The median (IQR) CD4 count was 446 (325-614) cells/mm3 and 208 (94.1%) of 221 patients tested had HIV-1 RNA <400 copies/mL. A total of 412 (85.8%) had detectable HCV RNA, at a median (IQR) of 6.2 (5.4-6.6) log10 IU/mL. Among 380 patients with HCV GT, 223 (58.7%) had GT1, 97 (25.5%) had GT3, 43 (11.3%) had GT6, eight (2.1%) had GT4, two (0.5%) had GT2, and seven (1.8%) had indeterminate GT. Of 222 patients with IL28B testing, 189 (85.1%) had rs12979860 CC genotype, and 199 (89.6%) had rs8099917 TT genotype. Of 380 patients with FibroScan® , 143 (37.6%) had no/mild liver fibrosis (F0-F1), 83 (21.8%) had moderate fibrosis (F2), 74 (19.5%) had severe fibrosis (F3), and 79 (20.8%) had cirrhosis (F4). One patient (0.3%) had FibroScan® failure. In conclusion, a high proportion of HIV-HCV-coinfected patients had chronic HCV infection. HCV GT1 was predominant, and 62% of patients had liver disease warranting prompt treatment (≥F2).

Smoking and projected cardiovascular risk in an HIV-positive Asian regional cohort.

OBJECTIVES: The aim of the study was to assess the prevalence and characteristics associated with current smoking in an Asian HIV-positive cohort, to calculate the predictive risks of cardiovascular disease (CVD), coronary heart disease (CHD) and myocardial infarction (MI), and to identify the impact that simulated interventions may have. METHODS: Logistic regression analysis was used to distinguish associated current smoking characteristics. Five-year predictive risks of CVD, CHD and MI and the impact of simulated interventions were calculated utilizing the Data Collection on Adverse Effects of Anti-HIV Drugs Study (D:A:D) algorithm. RESULTS: Smoking status data were collected from 4274 participants and 1496 of these had sufficient data for simulated intervention calculations. Current smoking prevalence in these two groups was similar (23.2% vs. 19.9%, respectively). Characteristics associated with current smoking included age > 50 years compared with 30-39 years [odds ratio (OR) 0.65; 95% confidence interval (CI) 0.51-0.83], HIV exposure through injecting drug use compared with heterosexual exposure (OR 3.03; 95% CI 2.25-4.07), and receiving antiretroviral therapy (ART) at study sites in Singapore, South Korea, Malaysia, Japan and Vietnam in comparison to Thailand (all OR > 2). Women were less likely to smoke than men (OR 0.11; 95% CI 0.08-0.14). In simulated interventions, smoking cessation demonstrated the greatest impact in reducing CVD and CHD risk and closely approximated the impact of switching from abacavir to an alternate antiretroviral in the reduction of 5-year MI risk. CONCLUSIONS: Multiple interventions could reduce CVD, CHD and MI risk in Asian HIV-positive patients, with smoking cessation potentially being the most influential.

Atherosclerotic cardiovascular disease screening and management protocols among adult HIV clinics in Asia.

OBJECTIVES: Integration of HIV and non-communicable disease services improves the quality and efficiency of care in low- and middle-income countries (LMICs). We aimed to describe current practices for the screening and management of atherosclerotic cardiovascular disease (ASCVD) among adult HIV clinics in Asia. METHODS: Sixteen LMIC sites included in the International Epidemiology Databases to Evaluate AIDS - Asia-Pacific network were surveyed. RESULTS: Sites were mostly (81%) based in urban public referral hospitals. Half had protocols to assess tobacco and alcohol use. Protocols for assessing physical inactivity and obesity were in place at 31% and 38% of sites, respectively. Most sites provided educational material on ASCVD risk factors (between 56% and 75% depending on risk factors). A total of 94% reported performing routine screening for hypertension, 100% for hyperlipidaemia and 88% for diabetes. Routine ASCVD risk assessment was reported by 94% of sites. Protocols for the management of hypertension, hyperlipidaemia, diabetes, high ASCVD risk and chronic ischaemic stroke were in place at 50%, 69%, 56%, 19% and 38% of sites, respectively. Blood pressure monitoring was free for patients at 69% of sites; however, most required patients to pay some or all the costs for other ASCVD-related procedures. Medications available in the clinic or within the same facility included angiotensin-converting enzyme inhibitors (81%), statins (94%) and sulphonylureas (94%). CONCLUSION: The consistent availability of clinical screening, diagnostic testing and procedures and the availability of ASCVD medications in the Asian LMIC clinics surveyed are strengths that should be leveraged to improve the implementation of cardiovascular care protocols.

Modulation of fibronectin gene expression in chondrocytes by viral transformation and substrate attachment.

Chicken vertebral chondrocytes, which normally grow in suspension, synthesize large amounts of cartilage extracellular matrix proteins, but little fibronectin. We have analyzed the effects of both substrate attachment and transformation with a temperature-sensitive mutant of Rous sarcoma virus on fibronectin gene expression in these cells. Our experiments show that viral transformation increases fibronectin synthesis to a greater extent than substrate attachment. Furthermore, transformed chondrocytes have lost the ability to decrease fibronectin synthesis in response to suspension culture, suggesting that transformation alters the normal attachment-responsive control of fibronectin gene expression. Finally, infected substrate-attached chondrocytes shifted to the nonpermissive temperature for transformation use fibronectin RNA more efficiently in protein synthesis than cells grown under the other conditions, suggesting for the first time a role for translational control of fibronectin gene expression.

Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells.

Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen.

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast-specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole-like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.

Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src.

A culture system was developed which permitted the differentiation of chicken lens epithelial cells to lentoid bodies which contained several cell layers, accumulated high levels of delta-crystallin, and produced extensive gap junctions. This differentiation process was prevented when the cells were infected with a temperature-sensitive src mutant of Rous sarcoma virus and maintained at the permissive temperature. These transformed cells continued to proliferate and also synthesized the major lens gap junction protein, MP28, at near-normal rates. However, this MP28 was not assembled to produce gap junctions. Cultures shifted to the nonpermissive temperature formed lentoid bodies similar to those in uninfected lens cultures, including the establishment of gap junctions containing MP28.

Expression of the Rous sarcoma virus src gene in avian macrophages fails to elicit transformed cell phenotype.

Infection of avian macrophages with Rous sarcoma virus does not induce any changes in the morphology, growth behavior, or expression of macrophage-specific proteins. The absence of cellular transformation does not result from a block in the synthesis of viral proteins, since infectious viruses are released from a majority of cells in the culture. In this report, we examine the synthesis, processing, and functional activity of pp60src in Rous sarcoma virus-infected macrophages to determine whether the absence of transformation is due to an alteration in the functional expression of pp60src. Although the absolute level of pp60src was reduced compared with fibroblasts, the protein exhibited the same phosphorylation pattern and subcellular distribution and was able to phosphorylate immunoglobulin in the immune complex-protein kinase assay. These results imply that the failure of Rous sarcoma virus to transform macrophage may be due to a restriction in the cellular response to a functional src protein, perhaps due to the absence of cellular products which are essential for mediating pp60src-induced transformation.

myc and src oncogenes have complementary effects on cell proliferation and expression of specific extracellular matrix components in definitive chondroblasts.

The effects of the avian viral oncogenes src and myc were compared for their ability to alter the differentiated phenotype and the proliferative capacity of definitive chondroblasts. As previously demonstrated, viruses carrying the src oncogene suppressed the synthesis of the chondroblast-specific products, type II collagen and cartilage-specific sulfated proteoglycan. In contrast, infection with MC29 and HB1 viruses, which carry the myc oncogene, did not suppress the synthesis of these normal differentiated cell products, but the infected cells exhibited an increased proliferative potential. The MH2 virus, which carries both the myc and mil oncogenes, both induced the suppression of these chondroblast-specific products and increased cell proliferation. The implications of these results for cooperation between oncogenes and the multi-oncogene models for neoplastic transformation are discussed.

Transformation of chicken embryo fibroblasts by v-src uncouples beta1 integrin-mediated outside-in but not inside-out signaling.

Adhesion of cells to extracellular matrix is mediated by integrin family receptors. The process of receptor-ligand binding is dependent on metabolic energy and is regulated by intracellular signals, termed inside-out signals. The strength of the initial alpha5beta1-mediated adhesion of v-src-transformed chicken embryo fibroblasts (v-srcCEF) was similar to that of normal CEF. A chemically cross-linked fibronectin substrate was able to restore cell spreading and the ability of v-srcCEF to assemble a fibronectin matrix. Over time, v-srcCEF showed decreased adhesion due to the reduction of alpha5beta1-fibronectin bonds consequent on the reduction of substrate-bound fibronectin due to the secretion of proteases by v-srcCEF. Excess synthesis of hyaluronic acid by v-srcCEF also reduced the alpha5beta1-fibronectin bonds and contributed to cell detachment at later times in culture. Thus, the adhesion defects were not due to a failure of alpha5beta1 function and adhesion of the v-srcCEF was alpha5beta1 dependent. Integrin-mediated adhesion also produces signals that affect cell proliferation and cell differentiation. An early consequence of these "outside-in" signals was the phosphorylation of FAK Y397 in direct proportion to the number of alpha5beta1-fibronectin bonds formed. In contrast, v-srcCEF had an increased level of phosphorylation on five different tyrosines in FAK, and none of these phosphorylation levels were sensitive to the number of alpha5beta1-fibronectin bonds. In the absence of serum, CEF proliferation was sensitive to changes in alpha5beta1-mediated adhesion levels. Transformation by v-src increased the serum-free proliferation rate and made it insensitive to alpha5beta1-mediated adhesion. Thus, the v-srcCEF were insensitive to the normal outside-in signals from alpha5beta1 integrin.

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.

The response of chicken embryo dermal fibroblasts to cytochalasin B is altered by Rous sarcoma virus-induced cell transformation.

The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborized or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transformed cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp60src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.

Control of types I and II collagen and fibronectin gene expression in chondrocytes delineated by viral transformation.

We have analyzed the effects of transformation by Rous sarcoma virus on expression of types I and II collagen and fibronectin genes in vertebral chondrocytes and compared them with expression of these genes in skin fibroblasts. Transformed chondrocytes display a dramatically decreased amount of type II collagen RNA, which can account fully for the decreased synthetic rate of this protein. Paradoxically, these cells also display greatly increased amounts of type I collagen RNAs, which are translated efficiently in vitro, but not in the intact cells. We show here that the type I collagen RNAs in transformed chondrocytes are nearly indistinguishable from those found in skin fibroblasts, and that they clearly differ from the type I collagen RNAs found in normal chondrocytes. Transformed chondrocytes also display an increased amount of fibronectin RNAs, which can account fully for the increased synthetic rate of this protein. Thus, the effects of transformation by Rous sarcoma virus on type I collagen and fibronectin RNAs in chondrocytes are the opposite of those observed in fibroblasts, which display decreased amounts of these three RNAs. These data indicate that the effects of transformation on the genes encoding type I collagen and fibronectin must be modulated by host cell-specific factors. They also imply that the types I and II collagen genes may be regulated by different mechanisms, the type I genes being controlled at both transcriptional and posttranscriptional levels, and the type II gene being controlled primarily at the transcriptional level.

Modulation of cell proliferation and differentiation through substrate-dependent changes in fibronectin conformation.

Integrin-mediated cell adhesion to extracellular matrices provides signals essential for cell cycle progression and differentiation. We demonstrate that substrate-dependent changes in the conformation of adsorbed fibronectin (Fn) modulated integrin binding and controlled switching between proliferation and differentiation. Adsorption of Fn onto bacterial polystyrene (B), tissue culture polystyrene (T), and collagen (C) resulted in differences in Fn conformation as indicated by antibody binding. Using a biochemical method to quantify bound integrins in cultured cells, we found that differences in Fn conformation altered the quantity of bound alpha5 and beta1 integrin subunits but not alphav or beta3. C2C12 myoblasts grown on these Fn-coated substrates proliferated to different levels (B > T > C). Immunostaining for muscle-specific myosin revealed minimal differentiation on B, significant levels on T, and extensive differentiation on C. Differentiation required binding to the RGD cell binding site in Fn and was blocked by antibodies specific for this site. Switching between proliferation and differentiation was controlled by the levels of alpha5beta1 integrin bound to Fn, and differentiation was inhibited by anti-alpha5, but not anti-alphav, antibodies, suggesting distinct integrin-mediated signaling pathways. Control of cell proliferation and differentiation through conformational changes in extracellular matrix proteins represents a versatile mechanism to elicit specific cellular responses for biological and biotechnological applications.

Activation of alpha(v)beta3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of beta3.

Integrin receptors serve as mechanical links between the cell and its structural environment. Using alpha(v)beta3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between alpha(v)beta3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the alpha(v)beta3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the beta3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of alpha(v)beta3 integrin.

Selective effect of Rous sarcoma virus src gene expression on contractile protein synthesis in chick embryo myotubes.

Myogenic precursor cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and maintained at the permissive temperature for transformation. Following subculture, a population of cells was produced which failed to differentiate at the permissive temperature but produced a high proportion of myotubes in sister cultures shifted to the nonpermissive temperature. The myotube-containing cultures were further enriched for this cell type by the addition of 1-beta-D-arabinofuranosylcytosine to kill replicating mononucleated cells. This myotube population was suitable for testing the effect of viral-transforming gene expression in a postmitotic, terminally differentiated cell which expresses relatively low levels of the cellular homologue of the viral-transforming gene and is resistant to infection by the virus. The consequence of shifting these Rous sarcoma virus-infected myotube cultures to the permissive temperature was assessed in terms of protein synthesis. The total rate of incorporation of exogenous radioactive leucine, supplied at a concentration which saturated the intracellular pool, was similar between cultures held at the two temperatures, suggesting that the absolute rate of total protein synthesis was not affected by expression of viral-transforming gene. In contrast, the rate of synthesis of eight proteins the expression of which is specific for myotubes was suppressed reversibly. The rate of synthesis of five other proteins which are not selectively concentrated in myotubes was either unaffected or stimulated. Thus. the expression of viral-transforming gene in myotubes leads to differential effects on developmentally regulated proteins without inducing several properties of the transformed state classically observed for fibroblastic cells.

Aggregation of platelets and cell membrane vesiculation by rat cells transformed in vitro by Rous sarcoma virus.

Primary rat embryo cells and established normal rat kidney cells transformed in vitro by Rous sarcoma virus induced the aggregation of rat platelets in vitro. The aggregating activity was shown to be specific for the transformed cells and was absent in the normal parent cells. The aggregation reaction is accompanied by the release of serotonin from the platelets. Further analysis and purification of this activity from the transformed cells demonstrated that the activity is shed from the cells growing in culture and is associated with membrane vesicles of heterogenous size. The normal cells also produced vesicles in culture; however, the level of vesicle productio was less than that from transformed cells, and the platelet aggregation and serotonin release activities were greatly reduced or absent in these vesicles.

Expression of v-src alters the expression of myogenic regulatory factor genes.

Inactivation of v-src in transformed chicken myoblasts induced changes in expression of the myogenic regulatory factors (MRF's), MyoD, myogenin and Myf5. Both MyoD and Myf5 were expressed in the transformed cells. The inactivation of v-src by a temperature up-shift resulted in the loss of Myf5 expression, a transient increase in MyoD, followed by a rise in myogenin mRNA levels. The increase in transcripts for the later muscle proteins, myosin heavy and light chains, alpha-actin and troponin T followed the sequence of changes in MRF expression suggesting that their induction was the result of altered MRF expression. Thus, v-src appears to operate through effects on MRF's and not directly on the expression of the late muscle-specific proteins. When v-src was reactivated in mature myotubes by a temperature down-shift, the pattern of expression was quite different, the late muscle-specific transcripts decreased before the loss of myogenin transcripts and before the rise of Myf5 transcripts.

Phosphorylation of integrin in differentiating ts-Rous sarcoma virus-infected myogenic cells.

The differentiation of primary myogenic cultures requires the attachment of the cells to an extracellular matrix substrate using an integrin family receptor. These integrin receptors can be phosphorylated on both their alpha and beta chains, and it has been postulated that phosphorylation regulates the receptor function. Quail myogenic clones transformed with ts-LA24A differentiated into mature myotubes following a temperature shift to nonpermissive temperature which inactivates the viral src kinas. Phosphorylation of integrin beta-1 chain and of at least one alpha chain was detected on both serine and tyrosine. An additional alpha chain(s) with a mobility similar to alpha 5 was not phosphorylated at either temperature. Following the induction of differentiation by a temperature shift, there was a marked decrease in integrin phosphorylation of both alpha and beta integrin chains. This decrease was more prominent for serine than for tyrosine, suggesting that src could not be the only kinase involved. The drop in integrin phosphorylation correlated with the initiation of differentiation, suggesting that integrin phosphorylation could be at least part of the mechanism by which myogenic differentiation is blocked by v-src.