Cell adhesion-dependent differences in endogenous protein phosphorylation on the surface of various cell lines

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [gamma-32P]ATP and [gamma-32P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.     

Cooperative roles of PAK1 and filamin A in regulation of vimentin assembly and cell extension formation

The formation of extensions in cell migration requires tightly coordinated reorganization of all three cytoskeletal polymers but the mechanisms by which intermediate filament networks interact with actin to generate extensions are not well-defined. We examined interactions of the actin binding protein filamin A (FLNA) with vimentin in extension formation by fibroblasts. Knockdown (KD) of vimentin in fibroblasts reduced the lengths of cell extensions by 50% (p < 0.001). After cell binding to fibronectin, there was a time-dependent increase of phosphorylation of serine 39, 56 and 72 in vimentin, which was associated with vimentin filament assembly. Of the FLNA-interacting kinases that could phosphorylate vimentin, we focused on PAK1, which we found by reciprocal immunoprecipitation associated with FLNA. Enzyme inhibitor studies and siRNA KD demonstrated that PAK1 was required for vimentin phosphorylation and formation of cell extensions. In sedimentation assays, vimentin was exclusively detected in the insoluble pellet fraction of cells expressing FLNA while in FLNA KD cells there was increased vimentin in the supernatants of FLN KD cells. Compared with wild type, FLNA KD cells showed loss of phosphorylation of serine 56 and 72 in vimentin and reduced numbers and lengths of cell extensions by >4-fold. We suggest that the association of PAK1 with FLNA enables vimentin phosphorylation and filament assembly, which are important in the development and stabilization of cell extensions during cell migration.     

Interferon gamma-mediated growth regulation of epithelial cells

Interferon gamma (IFNgamma) is known to inhibit proliferation of certain transformed cell lines. Recently, we have demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNgamma (Burova et al., 2007) and provided direct evidence for the dependence of IFNgamma-induced EGFR transactivation upon EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNgamma on human epithelial cells lines A431 and HeLa which express high levels of EGFR, as well as HEK293, which expresses low levels of EGFR. We characterized the IFNgamma-induced changes in these cells by studying cell growth, the cell cycle and induction of apoptosis. The response to IFNgamma differed in the tested cell lines: cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as shown by cell counts and MTT. The cell cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNgamma. In contrast, IFNgamma treatment did not alter distribution by cell cycle phases in HEK293. Our results indicate that IFNgamma exhibit an antiproliferative effect depending on the increased expression of EGFR in A431 and HeLa cells. Further, it was demonstrated that IFNgamma induced the caspase 3 activation in A431 cells, suggesting an involvement of active caspase 3 in IFNgamma-induced apoptosis.     

Interferon-γ effect on growth regulation of cells with different levels of EGF receptor expression

Interferon gamma (IFNγ) is known to inhibit the proliferation of some transformed cell lines. Recently, we demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNγ (Burova et al., 2007) and provided direct evidence for the dependence of IFNγ-induced EGFR transactivation on the EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNγ on human epithelial cell lines—A431 and HeLa that express high levels of EGFR, as well as HEK293 that expresses low levels of EGFR. To characterize the IFNγ-induced changes in these cells, we studied cell growth, the cell cycle, and induction of apoptosis. The response to IFNγ differed in the compared cell lines; cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as was shown by the cell count and MTT. The cell-cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNγ. On the contrary, in HEK293 cells, the IFNγ treatment did not alter distribution by cell cycle phases. Our results indicate that IFNγ produces an antiproliferative effect that depends on the increased expression of EGFR in A431 and HeLa cells. Furthermore, it was demonstrated that IFNγ induced the caspase 3 activation in A431 cells, which suggests the involvement of active caspase 3 in the IFNγ-induced apoptosis.     

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.     

Induction of stress response and differential expression of 70 kDa stress proteins by sodium fluoride in hela and rat brain tumor 9l cells

We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-κB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed. J. Cell. Biochem. 69:221–231, 1998. © 1998 Wiley-Liss, Inc.     

Taxol induces concomitant hyperphosphorylation and reorganization of vimentin intermediate filaments in 9l rat brain tumor cells

Taxol, a microtubule stabilizing agent, has been extensively investigated for its antitumor activity. The cytotoxic effect of taxol is generally attributed to its antimicrotubule activity and is believed to be cell cycle dependent. Herein, we report that taxol induces hyperphosphorylation and reorganization of the vimentin intermediate filament in 9L rat brain tumor cells, in concentration- and time-dependent manner. Phosphorylation of vimentin was maximum at 10⁻⁶ M of taxol treatment for 8 h and diminished at higher (10⁻⁵ M) concentration. Enhanced phosphorylation of vimentin was detectable at 2 h treatment with 10⁻⁶ M taxol and was maximum after 12 h of treatment. Taxol-induced phosphorylation of vimentin was largely abolished in cells pretreated with staurosporine and bisindolymaleimide but was unaffected by H-89, KT-5926, SB203580, genistein, and olomoucine. Thus, protein kinase C may be involved in this process. Hyperphosphorylation of vimentin was accompanied by rounding up of cells as revealed by scanning electron microscopy. Moreover, there was a concomitant reorganization of the vimentin intermediate filament in the taxol-treated cells, whereas the microtubules and the actin microfilaments were less affected. Taken together, our data demonstrate that taxol induces hyperphosphorylation of vimentin with concomitant reorganization of the vimentin intermediate filament and that this process may be mediated via a protein kinase C signaling pathway. J. Cell Biochem. 68:472–483, 1998. © 1998 Wiley-Liss, Inc.     

PCNA/cyclin expression and BrdU uptake define different subpopulations in different cell lines.

Monoclonal antibodies (MAb) to a 36 KD protein, proliferating cell nuclear antigen (PCNA/cyclin), have been previously shown to be capable of identifying proliferating cells in vitro as well as in alcohol-fixed, paraffin-embedded tissue specimens. The routine use of these anti-PCNA/cyclin MAb in investigative studies and in diagnostic pathology requires a clearer understanding of the distribution of PCNA/cyclin in the different cell populations found in tissue specimens. We therefore compared the ability of MAb to three nucleus-associated proliferation markers (MAb 19A2 to PCNA/cyclin; Ki-67 to an undefined proliferation-related marker; BU-1 to 5'-bromodeoxyuridine (BrdU) incorporated into DNA) to identify the proliferating cell fraction of various cells in vitro. The cell lines were chosen to represent a spectrum of proliferation rates (high to low) and cell lineage (mesenchymal vs epithelial, non-transformed vs malignant): (a) HeLa and A-431 (two malignant carcinoma cell lines with high proliferation rates); (b) SK-5 (a non-transformed fibroblast cell line with a low proliferation rate); (c) HUVE (a non-transformed human umbilical vein endothelial cell line with a low proliferation rate). Single and double labeling immunofluorescence studies were performed after uniform 1-hr incubations with BrdU. Comparison of the overlapping distributions of detectable PCNA/cyclin expression and BrdU incorporation demonstrated substantial qualitative and quantitative differences between the different cell lines. In two of the four cell lines (HeLa, A-431) the BrdU staining distributions formed inclusive subsets of the PCNA-positive cell populations. In the HUVE cell line the two populations overlapped incompletely. In one cell line, SK-5, the two populations were mutually exclusive. MAb Ki-67 demonstrated a pattern in the SK-5 cell line that was strongly predictive of PCNA positivity, while showing no associated patterns in the other three cell lines. We conclude that PCNA/cyclin expression detected by MAb may define different cell subpopulations in different cell types relative to those incorporating BrdU or expressing the target antigen for Ki-67. This has implications for the clinical study of mixed cell populations using these antibodies.     

Thrombin-induced vimentin phosphorylation in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells were stimulated with thrombin (1 unit/ml) for 15-30 s and then lysed with a solution of Triton X-100 containing [gamma-32P]adenosine triphosphate. Thrombin-stimulated human umbilical vein endothelial cells showed an enhanced incorporation of 32P into at least 12 different proteins as compared to control cells treated similarly. The observed enhanced phosphorylation required the active site of thrombin because diisopropylphosphoryl-thrombin had no effect on the level of phosphorylation. The molecular weight of one of the phosphoproteins was similar to that of the intermediate filament protein vimentin (55-60 kDa), a major protein in endothelial cells. This 59-kDa protein was Triton X-100-insoluble and reacted on a Western blot with antibody raised in guinea pig against Chinese hamster ovary cell vimentin. Addition of the anti-vimentin antibody to the thrombin-stimulated, phosphorylated lysate immuno-precipitated a single 32P-labeled protein (59 kDa). These results demonstrate that thrombin rapidly stimulates the phosphorylation of vimentin in cultured endothelial cells and links thrombin stimulation to the phosphorylation of a cytoskeletal protein.     

Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ-³²P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of ³²P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of ³²P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.     

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.     

Association of mRNA and eIF-2 alpha with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2 alpha to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2 alpha shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.     

Anti-phosphopeptide antibody, P-STM as a novel tool for detecting mitotic phosphoproteins: identification of lamins A and C as two major targets

A polyclonal, phospho-epitope-specific antibody (P-STM) was generated to detect the activated p21-activated kinase 2 (PAK2), based on the regulatory autophosphorylation site Thr(402) of PAK2 [Yu et al., 1998]. In this report, we show that this antibody can also recognize many phosphoproteins in mitotic HeLa and A431 cells. Signal of these phosphoproteins emerged after treating the cells with nocodazole and okadaic acid, and was highly detected in G2-M phase transition of HeLa cells released from double thymidine block. Immunofluorescence analysis revealed that P-STM strongly stained HeLa cells at prometaphase and metaphase, but not at interphase and anaphase. Interestingly, this staining pattern was almost identical to that obtained by staining with MPM2, a monoclonal antibody known to react with phosphoproteins in mitotic HeLa cells. However, the phosphoproteins detected by the two antibodies are quite different. Two-dimensional gel electrophoresis (2DE) and tryptic peptide fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry were employed to identify lamins A and C as two of the mitotic cell-specific phosphoproteins recognized by P-STM. Lamins A and C immunoprecipitated from nocodazole-treated cells, but not from untreated cells showed strong reactivity to P-STM, and this reactivity lost completely after protein phosphatase 2A treatment. In summary, our results show that P-STM represents a novel tool for detecting mitotic phosphoproteins, which are different from those recognized by MPM2, and that lamins A and C are the two prominent mitotic phosphoproteins detected by P-STM.     

Association of mRNA and eIF-2α with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2α to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2α shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.     

Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.     

Intracellular IL-1 receptor antagonist type 1 inhibits IL-1-induced cytokine production in keratinocytes through binding to the third component of the COP9 signalosome

The IL-1 receptor antagonist (IL-1Ra) exists in four isoforms, three of which lack signal peptides and are primarily intracellular proteins. The biologic roles of the intracellular isoforms of IL-1Ra have remained unknown. The objective of these studies was to determine whether the major intracellular isoform of IL-1Ra 18-kDa type 1 (icIL-1Ra1), mediated unique functions inside cells. A yeast two-hybrid screen with HeLa cell lysates revealed specific binding of icIL-1Ra1, and not of the other IL-1Ra isoforms, to the third component of the COP9 signalosome complex (CSN3). This binding was confirmed by Far Western blot analysis, sedimentation on a glycerol gradient, glutathione pull-down experiments, and coimmunoprecipitation. In addition to binding specifically to CSN3, icIL-1Ra1 inhibited phosphorylation of p53, c-Jun, and IkappaB by the crude CSN-associated kinase and of p53 by recombinant protein kinase CK2 and protein kinase D, both associated with CSN3. The biologic relevance of the interaction between icIL-1Ra1 and CSN3 was demonstrated in the keratinocyte cell lines KB and A431, both possessing abundant CSN3. A431 cells exhibited high levels of icIL-1Ra1 but lacked both detectable IL-1alpha-induced IL-6 and IL-8 production and phosphorylation of p38 MAPK. KB cells displayed the opposite pattern which was reversed after transfection with icIL-1Ra1 mRNA. Inhibition of CSN3 or of icIL-1Ra1 production through gene knockdown with specific small interfering RNA in A431 cells each led to an inhibition of IL-1alpha-induced IL-6 and IL-8 production. Thus, icIL-1Ra1 exhibits unique anti-inflammatory properties inside cells through binding to CSN3 with subsequent inhibition of the p38 MAPK signal transduction pathway.     

Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors

Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell-bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937.     

Protein phosphorylation in response to the tumor promoter TPA is dependent on the state of differentiation of muscle cells

We have shown previously (A. Sobel and A. H. Tashjian, Jr. (1983). J. Biol. Chem. 258, 10,312-10,324;A. Sobel and M.C. Boutterin (1985). Neurochem. Int. 7, 995-1006) that, in the pituitary-derived GH4C1 cells, thyrotropin-releasing hormone or the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) stimulates the phosphorylation of two sets of cytoplasmic proteins related to the regulation of prolactin synthesis and release, respectively. Interestingly, phosphoproteins with identical electrophoretic migration properties on two-dimensional gels were detected in cultured neonate or adult mouse muscle cells and in the L6 and C2 myogenic cell lines. In addition TPA, which is known to have many actions on muscle cell functions, proliferation, and differentiation, stimulated the phosphorylation of these same proteins in myoblasts in culture. After fusion of the proliferating myoblasts into differentiated myotubes, this TPA-induced stimulation was strongly reduced in normal muscle cell cultures where some mononucleate muscle and non-muscle cells remained present. It was totally abolished in the homogeneous L6 and C2 cell lines. These observations suggest that the same phosphoproteins may be related to the intracellular mechanisms involved in the transduction of extracellular regulatory signals in such distinct differentiated environments as those of pituitary and muscle cells. In muscle cells themselves, the regulation of the phosphorylation of these proteins is function of the cell's state of differentiation.     

Nuclear retinoic-acid-binding proteins and receptors in retinoic-acid-responsive cell lines

Nuclear retinoic-acid-binding activity and the expression of retinoic acid receptor mRNA (RAR-alpha and RAR-beta) were assayed in the F9 embryonal carcinoma, HeLa, HL-60 promyelocytic leukaemia and S91 melanoma cell lines. A 4-svedberg nuclear retinoic-acid-binding activity was detected in all 4 cell lines, but the levels in the HeLa and HL-60 cells were lower than in the F9 and S91 lines. RAR-alpha mRNA was expressed in all 4 cell lines, although at a very low level in S91 cells. Conversely, RAR-beta mRNA was expressed in S91 cells and, at a lower level, in F9 cells but was undetectable in HeLa and HL-60 cells. RAR-beta, transcribed and translated in vitro from the cloned cDNA coding region, sedimented at 4 S and this suggests that the 4-svedberg nuclear retinoic-acid-binding activity may represent the retinoic acid receptors.