Hodgkin's lymphoma cells exhibit high expression levels of the PICOT protein.
ABSTRACT PICOT was originally discovered as a protein kinase C (PKC) binding protein in human Jurkat T-lymphocytes in which it was found to modulate PKCtheta-dependent functions. In addition, RT-PCR analysis suggested the expression of PICOT in a wide range of organs and cell types, including cells that are devoid of PKCtheta. We aimed at analyzing the expression of the PICOT protein in mouse lymphoid organs, and to compare them with those of Jurkat T-lymphocytes and other cell lines. We also analyzed whether PICOT expression in T-lymphocytes is dependent on the presence of PKCtheta, and whether it correlates with cell growth rate. Western blot analyses demonstrated PICOT expression in all lymphoid organs and cell lines tested. In addition, similar expression levels were observed in lymphoid organs of wild-type and PKCtheta-null mice, suggesting that PICOT expression in T-lymphocytes is independent of PKCtheta. However, PICOT expression levels were higher in Jurkat T-lymphocytes and other lymphoma cell lines compared to freshly isolated lymphocytes, while T-lymphocyte mitogens, such as concanavalin A, increased PICOT expression concomitantly with the induction of a faster T-lymphocyte growth rate. Finally, immunohistochemistry of freshly-isolated lymph nodes from Hodgkin's lymphoma patients revealed significantly higher levels of PICOT in Hodgkin's cells, compared to the normal surrounding lymphocytes. The present results show a direct correlation between PICOT expression levels and increased cell growth, both in vitro and in vivo, and suggest that immunostaining of PICOT might be useful for in situ identification of transformed cells, such as those of Hodgkin's lymphoma.
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ABSTRACT: Members of the protein kinase C (PKC) family of serine/threonine kinases play a key role in regulating the differentiation and growth of diverse cell types and, to date, the cloning of seven mammalian PKC genes encoding eight distinct isoforms has been reported. Here we describe the molecular cloning and deduced primary structure of a cDNA encoding a novel PKC isoform, termed PKC theta, which was isolated in the course of attempts to identify PKC genes that are expressed selectively in hematopoietic cells. Degenerate oligonucleotide primers corresponding to conserved sequence motifs, which distinguish the PKC family from other protein kinases, were employed in polymerase chain reactions (PCR) to amplify partial core sequences of putative PKC genes from a human peripheral blood lymphocyte-derived cDNA library. DNA sequencing of selected clones revealed several PKC-related sequences, including one that, on the basis of sequence comparison with known PKC isoforms, represented a novel PKC isoform. The complete cDNA sequence was determined by anchored PCR cloning and sequencing the entire coding sequence, using cDNA derived from a human leukemic T cell line (Jurkat). Included within this approximately 2.7-kilobase pair cDNA is an open reading frame of 2,118 nucleotides encoding a putative 82-kDa protein. The deduced primary structure contains consensus sequences characteristic of protein kinase catalytic domains and, based on its amino acid sequence and domain structure, is a member of the PKC family. PKC theta displays the highest homology to PKC delta, lacks the Ca(2+)-binding C2 domain and, thus, belongs to the subfamily of Ca(2+)-independent PKC enzymes which also includes the delta, epsilon, zeta, and eta isoforms. RNase protection assays and semiquantitative PCR analysis indicated that, although PKC theta transcripts are expressed ubiquitously, the highest levels are found in hematopoietic tissues and cell lines, including T cells and thymocytes. In contrast, the expression levels in the brain and testes are considerably lower, and no transcripts were detected in several human carcinoma cell lines. A rabbit antiserum raised against a unique (V3 domain) bacterially expressed PKC theta fragment immunoprecipitated specifically an 82-kDa protein from Jurkat cell lysates. Thus, PKC theta represents an additional member of the PKC family, and its predominant expression in hematopoietic cells suggests that it may play a role in signal transduction and growth regulatory pathways unique to these cells.Journal of Biological Chemistry 04/1993; 268(7):4997-5004. · 4.65 Impact Factor
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ABSTRACT: A comparative analysis of protein kinase C-theta (PKCtheta) protein expression was performed in various mouse organs and tissues, freshly isolated populations of mouse and human hematopoietic cells, primary leukemias, and established cell lines of different histological origins. Results demonstrated a predominant expression of PKCtheta in lymphoid tissues and skeletal muscle. Expression levels of PKCtheta, as well as PKCalpha, delta, epsilon, zeta, and eta in the thymus, were not markedly changed during postnatal development. High levels of expression were observed in CD4(+) and CD8(+) single-positive T cells and CD4(+)CD8(+) double-positive thymocytes, while B cells were completely devoid of PKCtheta. PKCtheta was found also in platelets, but relatively low levels or no detection of PKCtheta expression were observed in neutrophils, monocytes, and macrophages. Highly proliferating leukemic T cells of established lines or primary tumors, but not freshly isolated resting peripheral blood T cells, exhibited high levels of membrane-bound PKCtheta. Increased proportions of PKCtheta in the particulate fraction was not restricted to malignant cells but correlated with the extent of proliferation of the T cells. Thus, human peripheral blood T cells that were induced to proliferate by exposure to mitogen and IL-2 expressed increased levels of PKCtheta in the particulate fraction. Significantly lower proportions of membrane-bound PKC were observed for five other isoenzymes expressed in T cells. The occurrence of PKCtheta in T, but not B, cells and its subcellular distribution in proliferating cells implicate PKCtheta in cellular mechanisms regulating the sustained proliferation of T cells.Cellular Immunology 06/1999; 193(2):185-93. · 1.74 Impact Factor
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ABSTRACT: Protein kinase C (PKC)theta is an established component of the immunological synapse and has been implicated in the control of AP-1 and NF-kappaB. To study the physiological function of PKCtheta, we used gene targeting to generate a PKCtheta null allele in mice. Consistently, interleukin 2 production and T cell proliferative responses were strongly reduced in PKCtheta-deficient T cells. Surprisingly, however, we demonstrate that after CD3/CD28 engagement, deficiency of PKCtheta primarily abrogates NFAT transactivation. In contrast, NF-kappaB activation was only partially reduced. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-trisphosphate generation and intracellular Ca2+ mobilization. Our finding suggests that PKCtheta plays a critical and nonredundant role in T cell receptor-induced NFAT activation.Journal of Experimental Medicine 07/2003; 197(11):1525-35. · 13.21 Impact Factor