Sherif Tawfic

Antisense oligonucleotides against protein kinase CK2‐α inhibit growth of squamous cell carcinoma of the head and neck in vitro

Human squamous cell carcinomas of the head and neck (SCCHN) overexpress the protein kinase CK2, and elevated CK2 activity correlates with aggressive tumor behavior and poor clinical outcome. We therefore investigated whether interference with CK2 expression would inhibit SCCHN cell growth in vitro.

Significance of protein kinase CK2 nuclear signaling in neoplasia.

Many stimuli play a role in influencing the structure and function of chromatin and nuclear matrix through post‐translational modifications of the component proteins in these dynamic structures. We propose that the protein serine/threonine kinase CK2 (formerly casein kinase II) is one such agent that is involved in signal transduction in the nuclear matrix and chromatin in response to a variety of stimuli. Protein kinase CK2 appears to undergo rapid modulations in its association with nuclear matrix and nucleosomes in response to mitogenic signals and is involved in the phosphorylation of a variety of intrinsic proteins in these structures depending on the state of genomic activity. In addition, its association or loss from the nuclear matrix may also influence the apoptotic activity in the cell. CK2 has been found to be dysregulated in virtually all the neoplasias examined and nuclear association appears to be an important facet of its expression in tumor cells. We hypothesize that CK2 provides a functional paradigm linking the nuclear matrix and chromatin structures. Identification of precise loci of action of CK2 in these structures and how they influence the morphological appearance of the nucleus under normal and abnormal growth conditions would be an important future direction of investigation. J. Cell. Biochem. Suppl. 35:130–135, 2000. Published 2001 Wiley‐Liss, Inc.

Role of protein phosphorylation in post-translational regulation of protein B23 during programmed cell death in the prostate gland.

Protein B23 is a nucleolar and nuclear matrix-associated phosphoprotein that is involved in ribosome synthesis. Its expression and phosphorylation in rat ventral prostate, an androgen target organ, are profoundly influenced by androgens. Induction of programmed cell death (apoptosis) in the prostatic epithelium by androgen deprivation in the animal induces an early decline in protein B23 in the absence of a corresponding loss of protein B23 mRNA. We have now demonstrated that prostatic nuclei retain the ability to transcribe the B23 mRNA and that a significant amount of this mRNA persists even after 7 days of androgen deprivation when >80% of the prostatic epithelial cells have undergone apoptosis. The B23 mRNA from these nuclei is also translatable in vitro. However, the majority of the B23 mRNA is associated with free and short-stretch polysomes, which may account for the castration-induced decline in synthesis of protein B23 in vivo. In addition, the mechanism of down-regulation of protein B23 in apoptotic prostatic cells appears to relate to two coordinate signals, which include loss of phosphorylation of the protein as well as the expression of a protease active toward dephosphorylated protein B23, under these conditions.

The pattern of CD10 expression in selected pathologic entities of the prostate gland

There is increasing evidence that neuropeptides, including bombesin, may influence growth, angiogenesis, invasiveness, and metastasis in prostate cancer. One of the molecules tightly involved in the regulation of neuropeptide activity is the integral membrane glycoprotein CD10, or neutral endopeptidase 24.11. The pattern of CD10 expression in hyperplastic and neoplastic conditions of the prostate gland has not been previously described. Immunohistochemical staining for CD10 and high-molecular-weight cytokeratin was performed on 92 cases of paraffin-embedded tissue from needle-core biopsy specimens and prostatectomy specimens. Normal and hyperplastic acini showed strong and distinct membrane (apical and intercellular) and cytoplasmic CD10 expression in basal and secretory cells. In contrast, no intercellular membrane or cytoplasmic staining of secretory cells was seen in any cases of adenocarcinoma with Gleason patterns 2 or 3. A subset of high-Gleason grade adenocarcinoma (patterns 4 and 5) displayed CD10 expression in the secretory cells; those cases shared a distinct morphological pattern. Prostatic intraepithelial neoplasia (PIN) showed consistent absence of intercellular membrane and cytoplasmic CD10 expression in the secretory cells, with preserved expression in basal cells. Interestingly, the basal cells in basal cell hyperplasia lacked CD10 expression, and no expression was noted in the secretory cells in all cases examined. Atrophic acini and those associated with acute and chronic inflammation retained CD10 expression. In conclusion, a consistent differential pattern of CD10 expression was seen in basal cell hyperplasia, PIN, and adenocarcinoma, suggesting a role for CD10 in the pathobiology of the prostate gland.

Role of protein kinase CK2 in phosphorylation of nucleosomal proteins in relation to transcriptional activity

Protein kinase CK2 undergoes rapid translocation to nuclear matrix and nucleosomes on androgenic stimulation of growth in prostatic epithelial cells. Further, CK2 appears to be regulated differentially in the transcriptionally active and inactive nucleosomes. We have investigated the role of CK2 in phosphorylation of nucleosome-associated proteins in the transcriptionally active and inactive nucleosomes that were isolated from ventral prostate subjected to different androgenic status in vivo. Proteins associated with these nucleosomes were phosphorylated via the intrinsic protein kinase activity, using [γ−32P] ATP in the absence and presence of GTP. Several proteins appear to be potential substrates for CK2 associated with the nucleosomes. Among them are proteins that are differentially associated with the transcriptionally active and inactive nucleosomes. Phosphorylation of several of these proteins is modulated depending not only on their sites of association (i.e., active vs. inactive nucleosomes) but also on the state of transcriptional activity. Differential phosphorylation of specific proteins by CK2 associated with the active and inactive nucleosomes may be pertinent to the process of transcription regulation. (Mol Cell Biochem 191: 135–142, 1999)

Association of protein kinase CK2 with nuclear matrix: Influence of method of preparation of nuclear matrix

Nuclear matrix (NM) plays roles of fundamental structural and functional significance as the site of replication, transcription, and RNA processing and transport, acting as an anchor or attachment site for a variety of enzymes and other proteins involved in these activities. We have previously documented that protein kinase CK2 translocates from the cytosol to the nucleus, where it associates preferentially with chromatin and NM, in response to certain growth stimuli. Considering that characteristics of the isolated NM can depend on the procedure employed for its isolation, we compared three standard methods for NM preparation to confirm the association of intrinsic CK2 with this structure. Our data suggest that the method used for isolating the NM can quantitatively influence the measurable NM‐associated CK2. However, all three methods employed yielded qualitatively similar results with respect to the stimulus‐mediated modulation of NM‐associated CK2, thus further supporting the notion that NM is an important site for physiologically relevant functions of CK2. In addition, core filaments and cytoskeleton that were isolated by two of the preparative methods had a small but significant level of associated CK2 activity. J. Cell. Biochem. 64:499–504.

Apoptosis and growth inhibition of head and neck tumor cell line induced by epidermal growth factor receptor tyrosine kinase inhibitor

Overexpression of the epidermal growth factor (EGF) receptor, a hallmark of aerodigestive squamous cell carcinoma of the head and neck (SCCHN), correlates with aggressive tumor behavior. There is evidence that SCCHN cells auto-activate their EGF receptors. The receptor has therefore attracted interest as a potential therapeutic target. We tested the in vitro therapeutic efficacy of PD153035--a potent, specific inhibitor of the tyrosine kinase intrinsic to the EGF receptor--by employing a well-characterized cell line derived from human gingival SCCHN. DNA-synthesis and cell number were assayed for growth-inhibitory effects, phosphorylation of the EGF receptor was quantitated by immunoblot, and cell apoptosis was detected by terminal deoxytransferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) in situ assay. PD153035, at nanomolar concentrations, inhibited autophosphorylation of the EGF receptor induced by EGF stimulation and the inhibition occurred in a dose-dependent manner. Under the same conditions, PD153035 inhibited cell growth, and induced apoptosis of SCCHN cells in vitro. We conclude that selective inhibition of the EGF receptor tyrosine kinase completely abolishes EGF receptor phosphorylation resulting from receptor stimulation, and results in growth inhibition and apoptosis of SCCHN cells in vitro. By inducing cytostasis and apoptosis, this new class of inhibitors may be of therapeutic value against SCCHN.

Modulation of nuclear matrix protein phosphorylation by histones: Possible involvement of NM-associated protein kinase CK2 activity †

Nuclear matrix (NM), a proteinaceous network of filaments, dictates nuclear morphology and the structure/function of DNA. Phosphorylation of NM proteins is a potential signal for regulating matrix functions. Histones also are intimately involved in DNA structure and transcription. Here, we report that various histones enhanced 32P incorporation into certain NM proteins. Modulation of NM protein phosphorylation by histones is mediated through regulation of protein kinase CK2, a messenger‐independent serine/threonine kinase, which is significantly associated with the NM. The stimulatory effect of histones was mitigated by prior incubation of histones with DNA in the reaction. Phosphorylation of NM proteins was extensively reduced when an excess of the CK2‐specific peptide substrate was included in the phosphorylation reaction as a competitor. Also, enhancement in the NM‐associated CK2 activity by histones was blocked by inhibitors of CK2. Histone H1 effect appeared to be mediated mainly by charge effect since a stretch of polylysine induced a similar effect. Various histones also differentially affected the autophosphorylation of NM‐associated CK2 subunits. This may contribute to the observed effects of histones on the NM, resulting in an enhancement and differential pattern of NM protein phosphorylation. Such a regional modification of NM protein phosphorylation might influence the nuclear functions that require histone displacement, namely, replication and transcription. J. Cell. Biochem. 72:242–250, 1999.

Successful Conservative Management of Bilateral Renal Mucormycosis

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