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
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclin T1 was recently identified, together with cdk9 (previously named PITALRE), as part of the TAK multiprotein complex, a co-factor targeted by the human immunodeficiency virus Type 1 (HIV-1) protein named Tat, suggesting a role for this complex in transcription elongation. Although studies on mRNA and protein expression have shown that cyclin T1 is ubiquitous in adult human tissues, no data have yet been reported regarding the expression of this protein in different cell lineages. Using a polyclonal antiserum raised against cyclin T1, we investigated the pattern of expression of this protein in adult human tissues by immunohistochemistry. Cyclin T1 was expressed ubiquitously, although different levels of expression were found in various organs. Some specialized tissues, such as blood, lymphoid tissues, and cells of connective tissue origin, showed high cyclin T1 expression. These specific expression patterns are only partially justified by some well-known specialized functions of cyclin T1 in certain cell types, such as its involvement in peripheral blood lymphocytes and monocyte differentiation. The high expression level found in other tissues suggests new possible roles for cyclin T1 in cell types other than those of lymphoid tissue. (J Histochem Cytochem 49:685692)
Key Words: cyclin T1, cyclin T2, polyclonal antibody, immunohistochemistry, human tissues, keratinocytes
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Precise regulation of the cell cycle is a fundamental requirement for homeostasis of the eukaryotic cell. During the past decade, scientists successfully delved into the molecular machinery devoted to the fine regulation of the cell cycle phases and identified and characterized several genes and gene products involved. A key role in this is played by cell cycle kinases (cdk), relatively small proteins with an apparent molecular mass between 33 and 43 kD whose activity is regulated by the arrangement in a multimeric complex with larger proteins, called "cyclins" after their cyclical expression and degradation during the cell cycle. Cyclins, a family of proteins, are the regulatory subunits of the multimeric complex whose catalytic activity is provided by the cdk moiety. Different cdk/cyclin complexes, formed with clear-cut timing throughout the cell cycle, together with their phosphorylation/dephosphorylation efficiently regulate the activity of the multimeric holoenzyme. Conversely, cdk/cyclin complexes are negatively modulated by the binding of a family of small proteins called cdk inhibitors (
Human cyclin T has an amino-terminal cyclin box motif sharing 39% identity with the human cyclin and a carboxy-terminal PEST sequence (aa 709726), which is frequently found in G1 cyclins to regulate their turnover by cellular ubiquitination and proteolysis pathways (8 kb) widely expressed in human tissues. A longer transcript,
9.5 kb, has been detected in peripheral blood lymphocytes (PBLs) and a shorter transcript,
3.0 kb, is found exclusively in the testis. In addition an abundant 3.5-kb transcript has been found only in the ovary, using a cDNA probe corresponding to the C-terminal region of the cyclin (
Studies on cyclin T1 mRNA and protein suggest its ubiquitous expression in human tissues. However, no data are yet available about the expression of the cyclin T1 gene product in particular cell types. We developed and characterized a specific polyclonal antiserum to describe the cyclin T1 immunohistochemical pattern of expression in several human tissues.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Normal Tissues and Cell Lines
Normal tissues from autopsy 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 were 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 MCF-7 (breast cancer) and mouse NIH-3T3 cells 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. Human HaCaT immortalized keratinocytes (a gift from Dr. A. Venuti; Istituto Regina Elena, Rome, Italy) were cultured and were induced to differentiate by Ca++ addition as previously reported (
Antisera
The rabbit polyclonal immune serum against cyclin T1 was produced by immunizing rabbits with a bacterially expressed glutathione S-transferase (GST)cyclin T1 fusion protein. Expression and purification of the fusion protein were done as previously reported (
The production of the antiserum against cyclin T2a is described in the accompanying report (
In Vitro TranscriptionTranslation and Immunoprecipitation Assay
One microgram of supercoiled plasmid encoding for human cyclin T1 and cyclin T2a were used to program a TnT rabbit reticulocyte lysate (Promega; Madison, WI) under control of the T7 polymerase in the presence of [35S]-methionine. Immunoprecipitation was carried out as described previously (
Western Blotting
Western blotting on cell lysates or cell fractions was performed as described previously (
Immunohistochemistry
Immunohistochemistry was carried out essentially as described previously (
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of a New Specific Antiserum for Cyclin T1
A polyclonal antiserum was produced by immunizing rabbits with bacterially-expressed GSTcyclin T1. The specificity of this antiserum was assessed by immunoprecipitation. As shown in Fig 1A, the antiserum recognized a specific band with an apparent molecular mass of 87 kD in 35S-labeled SAOS-2 and T98G cell lysates (Fig 1, Lanes a and c). Preimmune serum from the same rabbit was used as a control (Fig 1, Lanes b and d). To prove that the antiserum was specific only for cyclin T1, the in vitro translated products of both cyclin T1 (Fig 1, Lane a) and cyclin T2a (Fig 1, Lane b) were western blotted and assayed with the antiserum anti-cyclin T1. As shown in Fig 1B, no crossreaction was observed. An identical specificity was found for the antiserum against cyclin T2a, as shown in the accompanying article (
|
For Western blotting analysis, protein extracts from MCF-7, SAOS-2, and T98G cells were used. Cyclin T1 expression was easily detectable in all these human cell lines by use of the specific antiserum (Fig 2). By means of a binding assay utilizing the in vitro-translated forms of the cyclin T family members, a negligible crossreactivity with cyclin T2a was observed. In addition, immune crossreactivity with murine cyclin T1 was demonstrated positively using a NIH-3T3 cell lysate (not shown).
|
Expression Pattern of Cyclin T1 in Normal Human Tissues
We used immunohistochemistry to determine the amount and the localization of cyclin T1 in a panel of different human tissues. Specimens from different individuals were analyzed for each of the tissues examined. Cyclin T1 was expressed widely, although a different tissue distribution and/or level of expression was detected in the different organs examined (Table 1). Interestingly, the expression pattern of cyclin T2a, another partner of cdk9, almost completely overlapped the pattern described for cyclin T1 (
Expression of Cyclin T1 in Epithelia
In general, epithelial cells, from either simple or stratified epithelium, showed positive staining for cyclin T1. Interestingly, stratified epithelia, such as epidermis and mucosa of the oral cavity, esophagus, cervix, and vagina, expressed a higher level of this protein in the immature basal and suprabasal layers (Fig 3A). A very similar pattern of expression was found for cyclin T2a (Fig 3C). Fig 3B and Fig 3D show the negative controls, performed by substituting the primary antisera with the respective preimmune sera. In the skin, a low expression level for cyclin T1 was found in hair follicles, sebaceous glands, and sweat glands. Strong immunoreactivity was detected in the stratified columnar epithelia of trachea, bronchi, and adjacent glands (Fig 3E, arrow 1). Pneumocytes displayed a temperate nuclear immunoreactivity for the protein. A comparable pattern of expression was found for cyclin T2a (Fig 3F). Glandular breast epithelium showed intermediate levels of expression for cyclin T1.
|
In the gastrointestinal system, medium to low positive nuclear staining for cyclin T1 was found in salivary glands, in the epithelium of the stomach, and in the gallbladder epithelia. A low level of immunoreactivity was found in small and large intestine, esophagus, and liver, with the exception of ductal biliary cells, which displayed a medium level of cyclin T1 expression (Fig 3I). An analogous expression was found for cyclin T2a (Fig 3J). The esocrine portion of the pancreas showed a low level of expression, whereas the exocrine portion displayed a medium level of expression for cyclin T1.
In the urinary system, cyclin T1 was expressed at a low level in all kidney tubules, in the uroepithelium, and in the prostate, whereas the glomeruli showed an intermediate expression level for cyclin T1.
Cyclin T1 was expressed at a low level in thyroid, pituitary, hypophysis, and adrenal glands, where staining was evident both in the chromaffin and in the cortical portion.
In the reproductive system, cyclin T1 was expressed at a very low level in all organs studied.
Expression of Cyclin T1 in Cardiovascular and Connective Tissue
Cyclin T1 showed a peculiar expression pattern in mesenchymal cells. High immunoreactivity for cyclin T1 was observed in skeletal muscle cells and in myocardial cells (Fig 3G) in all specimens examined. An identical phenomenon was observed for cyclin T2a (Fig 3H). A high level also was found in adipocytes, chondrocytes, and endothelial cells (Fig 3E, arrow 2), whereas fibroblasts and smooth muscle cells displayed a very low level of expression for cyclin T1.
Expression of Cyclin T1 in Central and Peripheral Nervous Systems
Neurons from different areas of the brain, such as frontal cortex and midbrain, cells of the granular level of the cerebellum, and Purkinje cells exhibited an intermediate nuclear staining. Perineural and endoneural cells of peripheral nerves and ganglion cells showed low to undetectable levels of the protein. However, astrocytes, oligodendroglial, and microglial cells of brain tissue had a high level of expression of cyclin T1.
Expression of Cyclin T1 in Hematopoieic Cells
A high level of cyclin T1 was observed in T-cells and B-cells, both in blood films and in lymphoid tissues (Fig 3E, arrows 2 and 3), whereas monocytes, eosinophils, neutrophils, and basophils showed medium expression for cyclin T1.
To confirm by an alternative approach the expression pattern of cyclin T1, we performed Western blotting analysis on several human fresh tissues. In Fig 4 a representative panel, including skin (Fig 4, Lane a), skeletal muscle (Fig 4, Lane b) and large intestine (Fig 4, Lane c), is shown. These results were consistent with the immunohistochemical pattern of expression and were in good agreement with the Northern blotting analyses described previously (
|
Expression of Cyclin T1 in Differentiating Immortalized Keratinocytes (HaCaT Cells)
After Ca++ addition to the culture medium, HaCaT cells undergo modifications that recapitulate the keratinocyte differentiation program (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biochemical and immunoistochemical characterization of a new polyclonal antiserum raised against cyclin T1 are described in this study. By taking advantage of a large human tissue bank, the pattern of expression of cyclin T1 in normal human tissues was analyzed. It is important to note that the antiserum assayed against the denatured products of cyclin T1 and cyclin T2a was specific only for cyclin T1. For that reason, it can be assumed that the immunohistochemical results are specific for cyclin T1.
After examination of the immunohistochemical pattern of expression of cyclin T1 in several human tissues, it can be concluded that cyclin T1 has a widespread pattern of expression, which is consistent with previous data on the RNA and protein expression profile for this protein in human tissues (
Certain tissues, such as those of the endocrine and reproductive systems, showed low cyclin T1 expression. On the other hand, tissues of mesenchymal origin, such as connective tissue, skeletal muscle, and myocardial cells, adipocytes, chondrocytes, and endothelial cells, as well as blood and lymphoid tissues, exhibited high cyclin T1 expression levels. In the nervous system, the highest level of expression was found in oligodendroglia and microglia.
This peculiar pattern of expression might account for the distinct activities of this protein in selected tissues. It has recently been demonstrated that, in some cell lineages, the expression level of cyclin T1 is not growth- and/or cell cycle-regulated. There is an exception in regard to peripheral blood lymphocytes, in which expression levels and activity of the cyclin T1/cdk9 complex are upregulated during lymphoid cell activation, suggesting a role for the cyclin T1/cdk9 complex in T-cell activation and in monocyte differentiation (
When cyclin T1 expression in human tissues was compared with that of cdk9 (
An exhaustive knowledge of the differential cell- and tissue-specific pattern of expression of cyclin T1 in normal human tissues is an essential requirement for critical evaluation of the exact role played by this protein in cell homeostasis. Moreover, the observation that regulation of cyclin T1 expression is dependent on tissue-specific signaling pathways (
![]() |
Acknowledgments |
---|
Supported in part by AIRC and Ministero della Sanità grants to MGP, by an FIRC grant to ADL, and by NIH grants RO1 CA 60999-01A1 and PO1 NS 36466 to AG. AB is a recipient of an FIRC fellowship.
We thank Dr P.G. Natali (Regina Elena Institute; Rome, Italy) for useful suggestions and criticism, Dr A. Venuti (Regina Elena Institute; Rome, Italy) for the HaCaT cell line, and Dr J.J. Gartland (Thomas Jefferson University) for editing the manuscript.
Received for publication September 25, 2000; accepted January 10, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bagella L, MacLachlan TK, Buono RJ, Pisano MM, Giordano A, De Luca A (1998) Cloning of murine CDK9/PITALRE and its tissue-specific expression in development. J Cell Physiol 177:206-213[Medline]
Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR (1999) Recruitment of cyclin T1/P-TEFb to an HIV type I long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription. Proc Natl Acad Sci USA 96:7791-7796
Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761-771[Abstract]
Chaturvedi V, Qin JZ, Denning MF, Choubey D, Diaz MO, Nickoloff BJ (1999) Apoptosis in proliferating, senescent, and immortalized keratinocytes. J Biol Chem 274:23358-23367
De Luca A, Esposito V, Baldi A, Claudio PP, Fu Y, Caputi M, Pisano MM, Baldi F, Giordano A (1997) CDC2-related kinase PITALRE phosphorylates pRb exclusively on serine and is widely expressed in human tissues. J Cell Physiol 172:265-273[Medline]
De Luca A, Tosolini A, Russo P, Severino A, Baldi A, De Luca L, Cavallotti I, Baldi F, Giordano A, Testa JR, Paggi MG (2001) Cyclin T2a gene maps on human chromosome 2q21. J. Histochem Cytochem 49:693-697
Frangioni JV, Neel BG (1993) Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210:179-187[Medline]
Garriga J, Peng JM, Parreño M, Price DH, Henderson EE, Graña X (1998) Upregulation of cyclin T1/CDK9 complexes during T cell activation. Oncogene 17:3093-3102[Medline]
Gold MO, Yang X, Herrmann CH, Rice AP (1998) PITALRE, the catalytic subunit of TAK, is required for human immunodeficiency virus Tat transactivation in vivo. J Virol 72:4448-4453
Herrmann CH, Carroll RG, Wei P, Jones KA, Rice AP (1998) Tat-associated kinase, TAK, activity is regulated by distinct mechanisms in peripheral blood lymphocytes and promonocytic cell lines. J Virol 72:9881-9888
Lew J, Huang Q-Q, Qi Z, Winkfein RJ, Aebersold R, Hunt T, Wang JH (1994) A brain-specific activator of cyclin-dependent kinase 5. Nature 371:423-426[Medline]
MacLachlan TK, Sang N, Giordano A (1995) Cyclins, cyclin-dependent kinases and Cdk inhibitors: Implications in cell cycle control and cancer. Eukariotic Gene Expr 5:127-156
Paramio JM, Laín S, Segrelles C, Lane EB, Jorcano JL (1998) Differential expression and functionally co-operative roles for the retinoblastoma family of proteins in epidermal differentiation. Oncogene 17:949-957[Medline]
Peng J, Zhu Y, Milton JT, Price DH (1998) Identification of multiple cyclin subunits of human P-TEFb. Genes Dev 12:755-762
Ping W, Mitchell EG, Shi-Min F, Wolfgang HF, Katherine AJ (1998) A novel cdk9-associated C-Type cyclin interacts directly with Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451-462[Medline]
Ping YH, Rana TM (1999) Tat-associated kinase (P-TEFb): a component of transcription preinitiation and elongation complexes. J Biol Chem 274:7399-7404
Tsai L-H, Delalle I, Caviness VS, Jr, Chae T, Harlow E (1994) p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 371:419-423[Medline]
Wei P, Garber ME, Fang SM, Fischer WH, Jones KA (1998) A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA. Cell 92:451-462[Medline]
Wimmer J, Fujinaga K, Taube R, Cujec TP, Zhu YR, Peng JM, Price DH, Peterlin BM (1999) Interactions between Tat and TAR and human immunodeficiency virus replication are facilitated by human cyclin T1 but not cyclins T2a or T2b. Virology 255:182-189[Medline]
Yang X, Gold MO, Tang DN, Lewis DE, Aguilar-Cordova E, Rice AP, Herrmann CH (1997) TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines. Proc Natl Acad Sci USA 94:12331-12336
Yankulov K, Bentley D (1998) Transcriptional control: Tat cofactors and transcriptional elongation. Curr Biol 8:R447-449[Medline]