ARTICLE |
Correspondence to: Marco G. Paggi, Laboratory for Cell Metabolism and Pharmacokinetics, Center for Experimental Research, Regina Elena Cancer Institute, Via delle Messi d'Oro, 156, 00158 Rome, Italy. E-mail: paggi@ifo.it
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Summary |
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Cyclin T2a was recently identified as one of the regulatory subunits of the cdkcyclin complex P-TEFb, the most studied positive factor in the regulation of transcription elongation. By fluorescent in situ hybridization (FISH), the gene codifying for cyclin T2a has been mapped on human chromosome 2q21. This locus also has been linked to different forms of myopathy. By use of a new specific antiserum raised against cyclin T2a, the immunohistochemical pattern of expression of cyclin T2a in human tissues has been examined and compared to that of cyclin T1, described in the previous report. The observation that immunohistochemical expression of cyclin T2a was high in skeletal muscle cells, whereas it was undetectable in two cases of centronuclear myopathy, together with its chromosomal location, suggests an involvement of the cdk9cyclin T2a complex in this disease. (J Histochem Cytochem 49:693697, 2001)
Key Words: cyclin T2a, immunohistochemistry, chromosome 2q21, FISH, skeletal muscle, myopathy
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Introduction |
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TRANSCRIPTION ELONGATION in eukaryotic genes is a complex process that involves a number of regulatory factors. A common rate-limiting step in the transcription of inducible genes is the release of RNA polymerase II (RNAPII) molecules from stalled elongation complexes that accumulate shortly after the initiation of RNA synthesis (
Here we describe the chromosomal mapping of the human cyclin T2a gene and the characterization of a new polyclonal antiserum raised against its gene product. By use of this antiserum, the immunohistochemical pattern of expression of cyclin T2a in normal human tissues was examined and was compared with that of cyclin T1, described in the previous report (
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Materials and Methods |
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Chromosomal Mapping
Metaphase spreads from phytohemagglutinin-stimulated lymphocytes of a healthy female donor were prepared as described (
Normal Tissues and Cell Lines
Normal tissues from autopsy, as well as tissue from two patients with myopathy, were obtained from the Department of Surgical Pathology of the Second University of Naples, Italy. Tissues were formalin-fixed and paraffin-embedded. Representative sections of each specimen were stained with hematoxylineosin and examined by a pathologist to confirm the histological preservation of the microanatomic structure. For each tissue examined, at least three specimens from two different individuals were analyzed.
Human tumor cell lines SAOS-2 (osteosarcoma), T98G (malignant glioma), and HeLa (cervical carcinoma) were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in culture in Dulbecco's modified Eagle's medium (DMEM) complemented with 10% fetal calf serum at 37C in a 5% CO2-containing atmosphere.
C2C7 mouse muscle myoblasts were plated at 1 x 104 cells/cm2 and maintained as monolayers in DMEM with 4.5 g/liter glucose, 10% fetal bovine serum, 2 mM glutamine, 200 U/ml penicillin G, and 200 µg/ml streptomycin at 37C in a 5% CO2 atmosphere. Differentiation of C2C7 cells was induced after 2 days in culture by switching the growth medium from one containing 10% fetal bovine serum to one containing 2% heat-inactivated horse serum.
Antiserum
The rabbit polyclonal immune serum against cyclin T2a was produced by immunizing rabbits with a bacterially expressed glutathione-S-transferase (GST)cyclin T2a fusion protein. The construct comprised the stretch aa 440663 of the protein. Expression and purification of the fusion protein was done as previously reported (
In Vitro TranscriptionTranslation and Immunoprecipitation Assay
One microgram of supercoiled plasmid encoding for human cyclin T2a was used to program a TnT rabbit reticulocyte lysate (Promega; Madison, WI) under the control of the T7 polymerase in the presence of [35S]-methionine. Cell labeling with [35S]-methionine and immunoprecipitation were carried out as previously described (
Western Blotting
Western blotting on cell lysates or cell fractions was performed as described previously (
Immunohistochemistry
Immunohistochemistry was carried out essentially as previously described (
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Results |
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Human Cyclin T2a Gene Maps on Chromosome 2q21
Hybridization of the cyclin T2a cDNA probe to human chromosomes showed specific labeling on chromosome 2. Analysis of 22 metaphase spreads revealed that 52% (46/88) of all fluorescence signals hybridized to chromosome 2q. At least one signal specific for chromosome 2q was observed in 20 of the 22 metaphase spreads examined. The distribution of signals was as follows: two chromatids (nine cells), three chromatids (four cells), four chromatids (three cells). The hybridization efficiency was relatively high. All chromosome-specific signals were localized to 2q21 (Fig 1).
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Characterization of a New Specific Antiserum for Cyclin T2a
A polyclonal antiserum specific for the human cyclin T2a protein was produced in rabbits. The cDNA for cyclin T2a was translated in vitro and was precipitated using the rabbit polyclonal serum against the GSTcyclin T2a (Fig 2A, Lane c). The polyclonal serum effectively immunoprecipitated in vitro-translated cyclin T2a, whereas undetectable activity was found in the preimmune serum (Fig 2A, Lane b).
The specificity of the antiserum was also assayed by immunoprecipitation. Using cell lysates from [35S]-labeled T98G cells, we observed that cyclin T2a migrated with an apparent molecular mass of approximately 87 kD (Fig 2B, Lane b). The preimmune serum did not show any specific band (Fig 2B, Lane a).
To prove that the antiserum was specific only for cyclin T2a, the in vitro-translated products of both cyclin T1 (Fig 2A, Lane a) and cyclin T2a (Fig 2A, Lane b) were Western blotted and assayed with the antiserum anti-cyclin T2a. As shown in Fig 2C, no crossreaction was observed. An identical specificity was found for the antiserum against cyclin T1, as shown in the previous report (
For Western blotting analysis, protein extracts from SAOS-2, T98G, and HeLa cells were employed. Using the specific immune serum, cyclin T2a appeared as a doublet in all the cell lines tested (Fig 2D).
Expression Pattern of Cyclin T2a in Normal Human Tissues
We used immunohistochemical techniques to determine the localization of cyclin T2a in human adult tissues. At least three specimens from different individuals were analyzed for each of the tissues examined. Cyclin T2a protein was found to be ubiquitous, according to mRNA distribution (
Immunohistochemical Expression of Cyclin T2a in Two Cases of Myopathy
Myopathies are non-inflammatory primary degenerative muscle diseases that become symptomatic in infancy or childhood and are characterized by proximal or diffuse muscle weakness (
Our results showed that the human cyclin T2a gene mapped on this chromosomal locus and that cyclin T2a protein level was high in human adult skeletal muscle cells. Moreover, it has already been shown that there is increased cdk9cyclin T complex activity in some terminally differentiated tissues, and that it may be involved in muscle cell differentiation (
Drawing on this background, we investigated cyclin T2a immunohistochemical expression in two cases of centronuclear myopathy (
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Discussion |
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A new polyclonal antiserum raised against its protein product was produced to characterize cyclin T2a more accurately. Using this antiserum, we performed a biochemical and immunoistochemical characterization of cyclin T2a. Immunohistochemical analysis of cyclin T2a expression in a large human tissue bank shows that cyclin T2a is widely expressed in all cell types, even if higher levels are found in some terminally differentiated tissues such as muscle, blood, lymphoid tissue, and connective cells. This expression pattern is very similar to that described for cyclin T1 in the previous article (
We examined the possible specific role of cyclin T2a in terminally differentiated tissues, such as muscle. It is well known that, during myocyte differentiation, there is downregulation of cyclins D1, E, and A and upregulation of several cdk inhibitors which allow exit from cell cycle and expression of muscle-specific genes (
Because cyclin T2a gene was mapped on chromosome 2q21, a locus recently linked to different forms of myopathy (
We conclude that, apart from its involvement in transcription regulation, the cdk9cyclin T2a complex could have other functions. On the basis of high expression level in differentiated muscle tissues, we speculate that cdk9cyclin T2a activity could be involved in myogenic differentiation. The observation that immunohistochemical expression of cyclin T2a was undetectable in two patients with centronuclear myopathy suggests an involvement of the cdk9cyclin T2a complex in this disease. This is in agreement with the finding that the gene maps on a locus linked with different forms of myopathy and with its putative role in myogenic differentiation.
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Acknowledgments |
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Supported in part by AIRC and Ministero della Sanità grants to M.G.P., by a Second University of Naples and a CNR grant to A.D.L., by NIH grants RO1 CA 60999-01A1 and PO1 NS 36466 to A.G., by NCI grants CA45745 and CA06927 (Fox Chase Cancer Center), and by an appropriation from the Commonwealth of Pennsylvania to J.R.T. A.B. is recipient of an FIRC fellowship.
We thank Mr Tullio Battista (Regina Elena Institute; Rome, Italy) for his excellent technical assistance and Dr J.J. Gartland (Thomas Jefferson University; Philadelphia, PA) for editing the manuscript.
Received for publication September 25, 2000; accepted January 10, 2001.
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