Feasibility of immunocytochemical detection of tumor markers (XIAP, phosphohistone H1 and p63) in FNA cellblock samples from head and neck squamous cell carcinoma.

One of the major handicaps in contemporary clinical oncology is the inability to predict the responsiveness of any individual's malignancy to specific therapies. The purpose of this study was to test the feasibility of immunocytochemically detecting markers that may be affected by therapy or are predictive of therapeutic responsiveness, including phosphohistone H1 (anti-p-H1 MoAb 12D11) and X-linked inhibitor of apoptosis (XIAP) in small samples obtained via fine-needle aspiration (FNA) biopsy procedure, thus improving therapeutic monitoring. p63, a squamous stem cell regulatory protein, was also examined. These three markers were studied in FNA cell block samples of head and neck squamous cell carcinoma (HNSCC). Twenty-eight alcohol-fixed formalin-postfixed paraffin-embedded cell-block samples from FNAs of patients with HNSCC were subjected to antigen retrieval and then incubated with anti-XIAP, anti-p-H1, and anti-p63, and developed using EnVision-Plus reagents and diaminobenzidine as chromagen; Granular or heterogeneous cytoplasmic staining for XIAP and nuclear staining for p63 and p-H1 were considered positive. Among the 28 cases studied, the overall positive rates for XIAP, p-H1, and p63 were 60.7%, 96.4%, and 92.8%, respectively. The staining intensity for XIAP: + 70.6%, ++ 23.5%, +++ 0%, and ++++ 5.9%; for p-H1: + 48.1%, ++ 11.1%, +++37.0%, and ++++ 3.7%; and for p63: + 11.5%, ++ 23.1%, +++ 53.9%, and ++++ 11.5%. The expression of p-H1 and p63 appeared to be higher and stronger than that of XIAP in HNSCC. This study demonstrated the feasibility of monitoring expression of three tumor markers using FNA samples. p-H1 and XIAP may be useful for monitoring actions of cyclin-dependent kinase inhibitors, XIAP-lowering, and/or apoptosis-inducing drugs, respectively. Future studies will focus on the impact of therapies upon these staining profiles.

Immunohistochemical detection of GLUT1, p63 and phosphorylated histone H1 in head and neck squamous intraepithelial neoplasia: evidence for aberrations in hypoxia-related, cell cycle- and stem-cell-regulatory pathways.

AIM: Most epithelial malignancies are characterized by multistep progression from preinvasive/intraepithelial neoplasia to invasive malignancy. Detection and grading of early squamous intraepithelial neoplasia may at times be problematic. The aim of this study was to examine the ability of immunomarkers GLUT1, phospho-histone H1 and p63 to detect such early lesions. METHODS: Sections of formalin-fixed paraffin-embedded tissue from 27 cases of squamous intraepithelial neoplasia, 26 associated with invasive carcinoma, were immunostained with anti-p63 (4A4; Santa Cruz), anti-GLUT1 (Chemicon) and anti-phospho-histone H1 (monoclonal 12D11) and compared with normal, hyperplastic and immature squamous metaplastic epithelium. RESULTS: Normal epithelium displayed phospho-histone H1 in scattered parabasal cells; p63 in the basal one-quarter to one-half of epithelium; and GLUT1 negativity or weak/equivocal mid-epithelial GLUT1+ foci. In hyperplasia phospho-histone H1+ cells were also limited to the parabasal layer; p63 positivity was essentially identical to that in normal epithelium; GLUT1 characteristically stained basal cells in rete-like areas. p63 staining in squamous intraepithelial neoplasia (grade 1) was indistinguishable from normal epithelial staining; in contrast, squamous intraepithelial neoplasia (grade 3) was readily apparent, with up to full-thickness p63 positivity. Squamous intraepithelial neoplasia (grade 1) was readily distinguishable from normal epithelium with both phospho-histone H1 and GLUT1 immunostaining; both markers were detected in cell layers above the parabasal layer. With both, progressively higher cell layers stained in proportion to the severity of the intraepithelial neoplasia, up to full thickness positivity in grade 3 squamous intraepithelial neoplasia. Squamous metaplasia and grade 3 squamous intraepithelial neoplasia were not distinguishable with p63 (both showed full-thickness staining) but were readily distinguishable with GLUT1 and phospho-histone H1 stains. GLUT1 appeared to be the most sensitive marker for all grades of intraepithelial neoplasia. CONCLUSION: Altered expression of all three markers was a common finding in squamous intraepithelial neoplasia, hence, dysregulation of cell cycle-promoting cyclin-dependent kinases (phospho-histone H1), altered stem cell regulatory pathways (p63) and enhancement of hypoxia-sensing pathways (GLUT1) are all early alterations in the progression of squamous malignancy of head and neck origin. A panel of all three may be a useful means of detecting squamous intraepithelial neoplasia.

Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinization.

Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinizationAims : To examine invasive head and neck squamous carcinomas for expression of GLUT1, a glucose transporter and marker of increased glucose uptake, glycolytic metabolism and response to tissue hypoxia; p63, a p53 homologue that is a marker of the undifferentiated proliferative basaloid phenotype; and phospho-histone H1, a marker of activation of the cell cycle-promoting cyclin-dependent kinases 1 and 2. Methods : Routinely processed slides from 34 invasive squamous carcinomas, including 25 with intraepithelial components, were immunostained with anti-GLUT1 (Chemicon), anti-p63 (4A4, Santa Cruz), and antiphospho-histone H1 (monoclonal 12D11). Results : In keratinizing carcinomas, all three markers were most commonly immunodetected peripherally, with loss of expression in central keratinized zones. In contrast, in non-keratinizing carcinomas, p63 and phospho-histone H1 expression was most commonly observed throughout tumour nests and anti-GLUT1 stained in a pattern suggestive of hypoxia-induced expression ('antistromal' staining), in which cells at the tumour-stromal interface were GLUT1- and cells in central, perinecrotic zones showed progressive induction of GLUT1. Intraepithelial components also displayed basal and 'antibasal' GLUT1 staining patterns, homologous to the pro- and antistromal patterns in invasive carcinoma; basal patterns in intraepithelial lesions appeared to be more predictive of keratinizing invasive carcinoma and antibasal intraepithelial staining more predictive of non-keratinizing poorly differentiated carcinomas. Conclusions : Keratinizing and non-keratinizing squamous carcinomas differ in expression patterns of GLUT1, p63 and phospho-histone H1. In the former, all three markers were typically suppressed in conjunction with keratinization; in the latter, GLUT1 expression was more likely to occur in a hypoxia-inducible pattern and expression of p63 and phospho-histone H1 was unsuppressed. GLUT1 expression patterns in intraepithelial lesions may be predictive of the differentiation status of the associated invasive carcinoma.

Identification of the E2A gene products as regulatory targets of the G1 cyclin-dependent kinases.

The E2A gene products, E12 and E47, are multifunctional transcription factors that as homodimers regulate B cell development, growth, and survival. In this report, the E2A gene products are shown to be targets for regulation by the G1 cyclin-dependent kinases. Two novel G1 cyclin-dependent kinase sites are identified on the N-terminal domain of E12/E47. One site displays homology to a preferential D-type cyclin-dependent kinase site (serine 780) on the retinoblastoma susceptibility gene product (pRB) and, consistent with this homology, is more efficiently phosphorylated by cyclin D1-CDK4 than by the other cyclin-dependent kinases (CDK) that were tested. The second kinase site is phosphorylated by both cyclin D1-CDK4 and cyclin A/E-CDK2 complexes. Mutation studies indicated that phosphorylation of the cyclin D1-CDK4 site, or more potently, of both the cyclin D1-CDK4 and cyclin A/E-CDK2 sites, negatively regulates the growth suppressor function associated with the N-terminal domain of E12/E47. Transient expression studies showed that ectopic expression of cyclin D1 or E negatively regulates sequence-specific activation of gene transcription by E12/E47. Analysis of site mutants, however, indicated that inhibition of E12/E47 transcriptional activity did not require the N-terminal G1 cyclin-dependent kinase sites. Together, the results suggest that the growth suppressor and transcriptional activator functions of E12/E47 are targets for regulation by G1 cyclin-dependent kinases but that the mechanisms of regulation for each function are distinct.

SM-20 is a novel growth factor-responsive gene regulated during skeletal muscle development and differentiation.

SM-20 is a novel, evolutionarily conserved "early response" gene originally cloned from a rat aortic smooth muscle cell (SMC) cDNA library. SM-20 encodes a cytoplasmic protein, which is induced by platelet-derived growth factor and angiotensin II in cultured SMC and is upregulated in intimal SMC of atherosclerotic plaques and injured arteries. We have now examined SM-20 expression during differentiation of cultured skeletal myoblasts and during skeletal myogenesis in vivo. Low levels of SM-20 mRNA and protein were expressed in proliferating mouse C2C12 myoblasts. Differentiation by serum withdrawal was associated with a marked induction of SM-20 mRNA and the expression of high levels of SM-20 antigen in myotubes. The induction was partially inhibited by blocking differentiation with bFGF or TGFbeta. Similar results were obtained with the nonfusing mouse C25 myoblast line, suggesting that SM-20 upregulation is a consequence of biochemical differentiation and is fusion independent. During mouse embryogenesis, SM-20 was first observed at 8.5E in the dermomyotomal cells of the rostral somites. SM-20 expression progressed in a rostral to caudal pattern, with highest levels seen in the muscle primordia and mature muscles. SM-20 thus represents a novel intracellular protein that is regulated during skeletal muscle differentiation and development.

Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain.

The p42/44 mitogen-activated protein (MAP)-kinase cascade is a well-established signal transduction pathway that is initiated at the cell surface and terminates within the nucleus. More specifically, receptor tyrosine kinases can indirectly activate Raf, which in turn leads to activation of MEK and ERK and ultimately phosphorylation of Elk, a nuclear transcription factor. Recent reports have suggested that some members of p42/44 MAP kinase cascade can be sequestered within plasmalemmal caveolae in vivo. For example, morphological studies have directly shown that ERK-1/2 is concentrated in plasma membrane caveolae in vivo using immunoelectron microscopy. In addition, constitutive activation of the p42/44 MAP kinase cascade is sufficient to reversibly down-regulate caveolin-1 mRNA and protein expression. However, the functional relationship between the p42/44 MAP kinase cascade and caveolins remains unknown. Here, we examine the in vivo role of caveolins in regulating signaling along the MAP kinase cascade. We find that co-expression with caveolin 1 dramatically inhibits signaling from EGF-R, Raf, MEK-1 and ERK-2 to the nucleus. Using a variety of caveolin-1 deletion mutants, we mapped this in vivo inhibitory activity to caveolin-1 residues 32-95. Peptides derived from this region of caveolin 1 also inhibit the in vitro kinase activity of purified MEK-1 and ERK-2. Thus, we show here that caveolin-1 expression can inhibit signal transduction from the p42/44 MAP kinase cascade both in vitro and in vivo. Taken together with previous data, our results also suggest that a novel form of reciprocal negative regulation exists between p42/44 MAP kinase activation and caveolin-1 protein expression, i.e. up-regulation of caveolin-1 protein expression down-modulates p42/44 MAP kinase activity (this report) and up-regulation of p42/44 MAP kinase activity down-regulates caveolin-1 mRNA and protein expression.

Tissue factor expression in human arterial smooth muscle cells. TF is present in three cellular pools after growth factor stimulation.

Tissue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury.

Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo.

Caveolae are microdomains of the plasma membrane that have been implicated in organizing and compartmentalizing signal transducing molecules. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membrane in vivo. Recently, we and other laboratories have identified a family of caveolin-related proteins; caveolin has been re-termed caveolin-1. Here, we examine the cell-type and tissue-specific expression of caveolin-2. For this purpose, we generated a novel mono-specific monoclonal antibody probe that recognizes only caveolin-2, but not caveolins-1 and -3. A survey of cell and tissue types demonstrates that the caveolin-2 protein is most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts (L6, BC3H1, C2C12), fibroblasts, and 3T3-L1 cells differentiated to adipocytes. This pattern of caveolin-2 protein expression most closely resembles the cellular distribution of caveolin-1. In line with these observations, co-immunoprecipitation experiments with mono-specific antibodies directed against either caveolin-1 or caveolin-2 directly show that these molecules form a stable hetero-oligomeric complex. The in vivo relevance of this complex was further revealed by dual-labeling studies employing confocal laser scanning fluorescence microscopy. Our results indicate that caveolins 1 and 2 are strictly co-localized within the plasma membrane and other internal cellular membranes. Ultrastructurally, this pattern of caveolin-2 localization corresponds to caveolae membranes as seen by immunoelectron microscopy. Despite this strict co-localization, it appears that regulation of caveolin-2 expression occurs independently of the expression of either caveolin-1 or caveolin-3 as observed using two different model cell systems. Although caveolin-1 expression is down-regulated in response to oncogenic transformation of NIH 3T3 cells, caveolin-2 protein levels remain unchanged. Also, caveolin-2 protein levels remain unchanged during the differentiation of C2C12 cells from myoblasts to myotubes, while caveolin-3 levels are dramatically induced by this process. These results suggest that expression levels of caveolins 1, 2, and 3 can be independently up-regulated or down-regulated in response to a variety of distinct cellular cues.

MyoD functions as a transcriptional repressor in proliferating myoblasts.

The myogenic basic helix-loop-helix (myo-bHLH) proteins are a family of transcriptional regulators expressed in myoblasts and differentiated skeletal muscle. Ectopic expression of myo-bHLH regulators transdetermines some fibroblast cell lines into myoblasts, which exit the cell cycle and differentiate into skeletal muscle when cultured in low mitogen medium. While members of the myo-bHLH family have been shown to function as transcriptional activators in differentiating muscle, the molecular basis of their function in proliferating myoblasts has not been elucidated. In this report, we present evidence that MyoD functions as a transcriptional repressor in myoblasts. We show that transcription from a cyclin B1 promoter construct is repressed in proliferating myoblasts and that repression is mediated by a pair of MyoD binding sites. We also show that transcription from the cyclin B1 promoter is repressed in proliferating C3H10T1/2 cells by ectopic expression of MyoD. These results demonstrate that MyoD can repress transcription of specific genes in proliferating cells, a novel function that may be important to maintenance of the myogenic phenotype and to cell cycle regulation in myoblasts.

Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins.

Caveolae are microdomains of the plasma membrane that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal component of the caveolae membrane. Recently, we and others have identified a family of caveolin-related proteins; caveolin has been retermed caveolin-1. Caveolin-3 is most closely related to caveolin-1, but caveolin-3 mRNA is expressed only in muscle tissue types. Here, we examine (i) the expression of caveolin-3 protein in muscle tissue types and (ii) its localization within skeletal muscle fibers by immunofluorescence microscopy and subcellular fractionation. For this purpose, we generated a novel monoclonal antibody (mAb) probe that recognizes the unique N-terminal region of caveolin-3, but not other members of the caveolin gene family. A survey of tissues and muscle cell types by Western blot analysis reveals that the caveolin-3 protein is selectively expressed only in heart and skeletal muscle tissues, cardiac myocytes, and smooth muscle cells. Immunolocalization of caveolin-3 in skeletal muscle fibers demonstrates that caveolin-3 is localized to the sarcolemma (muscle cell plasma membrane) and coincides with the distribution of another muscle-specific plasma membrane marker protein, dystrophin. In addition, caveolin-3 protein expression is dramatically induced during the differentiation of C2C12 skeletal myoblasts in culture. Using differentiated C2C12 skeletal myoblasts as a model system, we observe that caveolin-3 co-fractionates with cytoplasmic signaling molecules (G-proteins and Src-like kinases) and members of the dystrophin complex (dystrophin, alpha-sarcoglycan, and beta-dystroglycan), but is clearly separated from the bulk of cellular proteins. Caveolin-3 co-immunoprecipitates with antibodies directed against dystrophin, suggesting that they are physically associated as a discrete complex. These results are consistent with previous immunoelectron microscopic studies demonstrating that dystrophin is localized to plasma membrane caveolae in smooth muscle cells.

Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin interacts directly with heterotrimeric G-proteins and can functionally regulate their activity. Recently, a second caveolin gene has been identified and termed caveolin-2. Here, we report the molecular cloning and expression of a third member of the caveolin gene gamily, caveolin-3. Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are approximately 65% identical and approximately 85% similar. A single stretch of eight amino acids (FED-VIAEP) is identical in caveolin-1, -2, and -3. This conserved region may represent a "caveolin signature sequence" that is characteristic of members of the caveolin gene family. Caveolin-3 mRNA is expressed predominantly in muscle tissue-types (skeletal muscle, diaphragm, and heart) and is selectively induced during the differentiation of skeletal C2C12 myoblasts in culture. In many respects, caveolin-3 is similar to caveolin-1: (i) caveolin-3 migrates in velocity gradients as a high molecular mass complex; (ii) caveolin-3 colocalizes with caveolin-1 by immunofluorescence microscopy and cell fractionation studies; and (iii) a caveolin-3-derived polypeptide functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins. Identification of a muscle-specific member of the caveolin gene family may have implications for understanding the role of caveolin in different muscle cell types (smooth, cardiac, and skeletal) as previous morphological studies have demonstrated that caveolae are abundant in these cells. Our results also suggest that other as yet unknown caveolin family members are likely to exist and may be expressed in a regulated or tissue-specific fashion.

Positive and negative regulation of D-type cyclin expression in skeletal myoblasts by basic fibroblast growth factor and transforming growth factor beta. A role for cyclin D1 in control of myoblast differentiation.

Differentiation of skeletal myoblasts in culture is negatively regulated by certain growth factors, including basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta). We investigated the effects of bFGF and TGF beta on D-type cyclin expression in skeletal myoblasts. When myoblasts were induced to differentiate in low mitogen medium, expression of cyclin D1 rapidly fell below detectable levels. In contrast, expression of cyclin D3 increased to levels exceeding those present in myoblasts. Expression of cyclin D1 was induced in myoblasts by bFGF and TGF beta (albeit with different kinetics for each factor), while induction of cyclin D3 expression was inhibited by these growth factors. Although these results are consistent with other reports showing induction of cyclin D1 by growth factors, induction of cyclin D3 expression during terminal differentiation of myoblasts and inhibition of this induction by growth factors is surprising. These results suggest that cyclin D3, previously thought to be only a positive regulator of cell cycle progression, may also function in the cellular context of terminal differentiated muscle. Stable expression of cyclin D1 from an ectopic viral promoter inhibits C2C12 myoblast differentiation, but only in those clones where the level of cyclin D1 expression does not significantly exceed that present in control myoblasts stimulated by bFGF. Together, these result suggest that cyclin D1 expression functions in the inhibition of myoblast differentiation by certain growth factors.

Ectopic expression of cyclin D1 prevents activation of gene transcription by myogenic basic helix-loop-helix regulators.

Activation of muscle gene transcription in differentiating skeletal myoblasts requires their withdrawal from the cell cycle. The effects of ectopic cyclin expression on activation of muscle gene transcription by myogenic basic helix-loop-helix (bHLH) regulators were investigated. Ectopic expression of cyclin D1, but not cyclins A, B1, B2, C, D3, and E, inhibited transcriptional activation of muscle gene reporter constructs by myogenic bHLH regulators in a dose-dependent manner. Ectopic expression of cyclin D1 inhibited the activity of a myogenic bHLH regulator mutant lacking the basic region protein kinase C site, indicating that phosphorylation of this site is not relevant to the mechanism of inhibition. Analysis of cyclin D1 mutants revealed that the C-terminal acidic region was required for inhibition of myogenic bHLH regulator activity, whereas an intact N-terminal pRb binding motif was not essential. Together, these results implicate expression of cyclin D1 as a central determinant of a putatively novel mechanism that links positive control of cell cycle progression to negative regulation of genes expressed in differentiated myocytes.

Structural and functional aspects of basic helix-loop-helix protein folding by heat-shock protein 90.

The assembly and folding of nascent polypeptides are mediated in vivo by molecular chaperones, many of which are members of the heat-shock family of proteins. We have shown previously that heat-shock protein 90 (HSP90) folds an inactive fraction of a recombinant basic helix-loop-helix (bHLH) protein generated in Escherichia coli (MyoD) into its active conformation. We show here that HSP90 also folds another bHLH protein (E12) and heterodimers of E12/MyoD into their active conformations. By purifying inactive heterodimers of E12/MyoD and subsequently rendering them active in binding DNA by treatment with HSP90, we show that one folding step mediated by HSP90 occurs after oligomerization of the bHLH protein monomers. A series of deletion mutants is used to identify the 48-amino acid region of HSP90 that confers bHLH folding activity, which lies near the COOH terminus. This region is required for activation of DNA binding of MyoD and E12 homodimers and E12/MyoD heterodimers.

Growth factors that repress myoblast differentiation sustain phosphorylation of a specific site on histone H1.

A monoclonal antibody (12D11) is presented that binds an epitope on histone H1 only when it is phosphorylated. Skeletal myoblasts, which are cultured in high mitogen medium to induce proliferation and inhibit differentiation, contain histone H1 reactive with monoclonal antibody 12D11, whereas differentiated myocytes and adult skeletal muscle do not. Phosphorylation of H1 at the 12D11 epitope, as assessed by antibody binding, is also induced and maintained in myoblasts cultured in low mitogen medium supplemented with transforming growth factor beta, which blocks differentiation but allows the cells to withdraw from the cell cycle (Olson, E., Sternberg, E., Hu, J., Spizz, G., and Wilcox, C. (1986) J. Cell Biol. 103, 1799-1805; Massague, J., Cheipetz, S., Endo, T., and Nadal-Ginard, B. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8206-8210). These observations suggest that phosphorylation of histone H1 at the 12D11 epitope is associated with the negative regulation of myoblast differentiation by growth factors.

Conformational activation of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of murine HSP90 (HSP84).

A murine cardiac lambda gt11 expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned. Both recombinant and native HSP90s were then found to rapidly convert a basic helix-loop-helix protein (MyoD1) from an inactive to an active conformation, as assayed by sequence-specific DNA binding. The conversion process involves a transient interaction between HSP90 and MyoD1 and does not result in the formation of a stable tertiary complex. Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion. HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells. These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.

Monoclonal antibodies identify a possible regulatory domain of MyoD1.

A panel of monoclonal antibodies (mAbs) to murine MyoD1 was generated. One set of mAbs is shown to react with epitope(s) in the cysteine/histidine-rich (C/H) region while another set is shown to react with epitope(s) in the C-terminal portion of MyoD1. One of the mAbs reactive with a C-terminal epitope sensitively detected MyoD1 in whole cell extracts by Western blotting. Time course studies of total protein accumulation during C2C12 myoblast differentiation revealed only subtle changes in the phosphorylation and quantity of MyoD1 protein present in C2C12 cells from induction to 120 hr after induction. These results suggest that modulation of MyoD1 protein or total phosphorylation levels is not tightly associated with the transition of undifferentiated myoblasts to differentiated myocytes. Monoclonal antibodies to the C-terminal epitope produced supershifted bands in gel retardation assays, indicating that these mAbs had no effect on DNA binding. Although the C/H region of MyoD1 does not participate in DNA binding, mAbs reactive with the C/H region neutralized this activity in gel retardation assays. These data suggest that the conserved C/H domain may serve to modulate MyoD1 DNA-binding activity by interacting with another regulator.

Four sarcomeric myosin heavy chain genes are expressed by human fetal skeletal muscle cells differentiating in culture.

Expression of four sarcomeric myosin heavy chain (MHC) genes was examined in continuously passaged human fetal (18-22 week) skeletal myoblasts and in myoblasts induced to differentiate by low mitogen medium. Although embryonic MHC mRNA predominated at all time points following induction, three additional MHC genes were expressed at lower levels. These consisted of perinatal, slow, and fast skeletal MHC genes. Temporal regulation of MHC gene expression was observed. In myoblasts and early induced cultures, embryonic and fast transcripts were detected, accompanied in later induced cultures by the accumulation of perinatal and slow MHC transcripts. In situ hybridization analysis of uninduced cells revealed that sarcomeric MHC transcripts originated from a small population of spontaneously fused multinucleated cells. Taken together, these observations demonstrate that human fetal myoblasts induced to differentiate in culture execute a developmental program that includes temporally regulated expression of four distinct sarcomeric MHC genes.

Infection and inhibition of differentiation of human fetal skeletal myoblasts by adenovirus.

The effects of adenovirus type 5 infection on the differentiation of cultured human skeletal myoblasts and of myoblast differentiation on the replication of adenovirus were investigated. Although infection of myoblasts concurrently with differentiation induction was inhibitory, myoblast differentiation was not impeded when infection was carried out 2 hr or later after induction. Similar studies conducted with E1A mutant viruses (dI312, pm975, and dI1500) revealed that complete inhibition was dependent on the product of 13 S E1A transcript expression, although partial inhibition could be induced by the 12 S product. Differentiation of myoblasts results in the generation of multinucleated myotubes and quiescent mononuclear cells. The three cell types (myoblasts, myotubes, mononuclear cells) were differentially permissive to adenovirus infection. The precursor myoblasts and the multinucleated myotubes were found to be permissive for adenovirus infection. The kinetics of their infection was delayed 24-48 hr relative to that of HeLa cells. Quiescent mononuclear cells in the differentiated myoblast cultures were found to be inefficient in supporting the production of adenovirus particles, despite the accumulation of adenovirus DNA and capsid proteins. Host protein synthesis in the three cell types also responded differently to adenovirus infection. In the multinucleated myotubes, host protein synthesis was potently inhibited by adenovirus at late times after infection, whereas it persisted in the proliferating myoblasts and quiescent mononuclear cells.

Novel NGF-induced proteins in PC12 cells: immunological evidence for their presence in brain nerve endings using a single monoclonal antibody.

A monoclonal antibody, S-11D9, detected a group of novel polypeptides whose expression was induced 6 hr after incubation of PC12 cells with nerve growth factor. The antigens also were visualized by immune precipitation and Western blotting in synaptic vesicles, clathrin-coated vesicles, and synaptic plasma membrane prepared from bovine brain. A large polypeptide was detected on the synaptic plasma membrane; an intermediate size protein was visualized on the plasma membrane and on both synaptic and clathrin-coated vesicles, while a smaller molecule was found only on brain Golgi-enriched membrane preparations. Immunofluorescence labeling with S-11D9 on nerve growth factor-stimulated PC12 cells showed the antigens distributed in the cytoplasm and concentrated in discrete areas on the tip of most neurites. The particular distribution of these proteins in vesicles and synaptic plasma membrane and the finding that the different antigens share a common epitope recognized by a single monoclonal antibody open the possibility that these molecules are related markers for organelle transport pathways in nerve cells.