Epstein-Barr virus renders the infected natural killer cell line, NKL resistant to doxorubicin-induced apoptosis.

We established two Epstein-Barr virus (EBV)-infected NKL sublines, which acquired stress resistant phenotype against DNA damage and starvation compared with EBV-negative NKL. EBV-rendered doxorubicin resistance at least partially through NF-kappaB activation and the resultant sustenance of antiapoptotic proteins including Bcl-X(L) and FLIP(L/S).

Infarction followed by hemorrhage in pituitary adenoma due to endocrine stimulation test.

A 63-year-old man, who presented with visual field loss due to pituitary tumor, received an intravenous bolus injection of thyrotropin and gonadotropin releasing hormones and insulin as a preoperative evaluation. He complained of severe headache and nausea 2 hours after injection. Emergent CT scan showed no evidence of intratumoral hemorrhage. The next day, his visual field became null. MR images revealed heterogeneous mixed intensity lesions. Under diagnosis of pituitary apoplexy, he underwent transsphenoidal tumor removal 30 hours after onset. Intraoperative and pathological findings showed tumor hemorrhage and adjacent necrotic change. Fourteen cases with sufficient clinical detail in the literature are reviewed: All of the cases had macroadenoma with suprasellar extension. Testing agents were gonadotropin and thyrotropin releasing hormones in 92.9% and 85.7% of cases, respectively. Headache was an initial symptom and started within two hours in all cases but one. Half of the cases showed no change on CT scan. However, tumor hemorrhage was evidenced in 92.9% of cases with or without necrosis due to ischemic change, intraoperatively or pathologically. It is speculated that pituitary apoplexy often starts with infarction possibly due to vasoactive effect of testing agents and later develops into hemorrhage. Therefore, it is necessary to observe patients closely at least a few hours after endocrine stimulation test, and MR imaging may make an earlier diagnosis for the pituitary apoplexy since CT scan often shows no density change in the pituitary adenoma.

Carcinoembryonic antigen-specific suicide gene therapy of cytosine deaminase/5-fluorocytosine enhanced by the cre/loxP system in the orthotopic gastric carcinoma model.

Tumor-specific gene delivery is crucial to achieving successful effects in suicide gene therapy. Carcinoembryonic antigen (CEA) promoter has been widely used for this purpose, but the expression level of tumor-specific promoters such as CEA promoter is generally low. In the previous study, we used the Cre/loxP system and showed that LacZ expression by the CEA promoter was remarkably enhanced and maintained its specificity using the Cre/loxP regulation system. In this study, the Cre/loxP system was first applied to augmentation of selective expression of the cytosine deaminase (CD) gene as a suicide gene therapy in CEA-producing cells. The double infection with AxCEANCre expressing Cre recombinase under the control of the CEA promoter and AxCALNLCD expressing the CD gene under the control of the CAG promoter by the Cre switching system rendered CEA-producing tumor cells 13-fold more sensitive to 5-fluorocytosine (5-FC) compared with the single infection with AxCEACD expressing CD gene driven by the CEA promoter. The therapeutic efficacy of the enhanced CD/5-FC suicide gene therapy was evaluated in orthotopic implantation models of human gastric carcinoma. Adenovirus vectors (1 x 10(9) plaque-forming units) were administered i.p. into mice three times, and then 5-FC was administered i.p. for the next 10 days. Tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD/5-FC were significantly reduced as compared with those in mice treated not only with Mock (AxCALacZ) but also with AxCEACD/5-FC (P < 0.0001). This beneficial effect on tumor burden was also reflected in the overall survival. The survival periods of the mice treated with AxCEANCre and AxCALNLCD/5-FC were longer than those of mice treated with Mock or AxCEACD/5-FC (P < 0.01). These results suggested that application of the Cre/loxP system could provide a new approach for enhanced selective suicide gene therapy of CD/5-FC for the treatment of advanced gastric carcinoma.

Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins.

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.

Demonstration of cyclin-dependent kinase inhibitory serine/threonine kinase in bovine thymus.

A synthetic peptide corresponding in sequence to residues 6-20 of p34cdc2, cdc2(6-20), and a substitution analogue, cdc2(6-20)F15K19 , which contains Thr-14 as the only phosphorylation target were used as substrates to identify a novel protein kinase in bovine thymus cytosol. The kinase catalyzed the phosphorylation of Thr-14 in both peptides and was purified extensively on the basis of its peptide phosphorylation activity. Upon SDS-polyacrylamide gel electrophoresis analyses, the purified samples consistently displayed a prominent 43-kDa protein band which could undergo in gel autophosphorylation, thus suggesting that this band represented the kinase protein. The suggestion was supported further by the observation that both cdc2(6-20) peptide phosphorylation and the autophosphorylation reaction of the 43-kDa protein were inhibited by millimolar concentrations of cAMP. The kinase was found to inactivate Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (Nclk), a heterodimer of Cdk5 and neuronal Cdk5 activator (Nck5a), under phosphorylation conditions. The phosphorylation of Nclk by the purified thymus kinase occurred on Cdk5. The monomeric form of Cdk5 was also phosphorylated by the kinase. Phosphoamino acid and phosphopeptide analysis of the phosphorylated Nclk revealed that Thr-14 of Cdk5 was the sole site of protein phosphorylation. The results suggest that this thymus kinase is a novel Cdk inhibitory protein kinase, distinct from the recently cloned dual functional and membrane-associated Cdc2 inhibitory kinase, Myt1 (Mueller, P. R., Coleman, T. R., Kumagai, A., and Durphy, W. G. (1995) Science 270, 86-90).

In vitro substrate specificity of protein tyrosine kinases.

Synthetic peptides such as P60src autophosphorylation site peptides and angiotensin are indiscriminately phosphorylated by protein tyrosine kinases. The observation has led to the general belief that protein tyrosine kinases are highly promiscuous, displaying little in vitro site specificity. In recent years, evidence has been accumulating to indicate that such a belief requires close examination. Synthetic peptides showing high substrate activity for specific groups of protein tyrosine kinases have been obtained. Systematic modification of certain substrate peptides suggests that kinase substrate determinants reside with specific amino acid residues proximal to the target tyrosine. A number of protein kinases have been shown to be regulated by tyrosine phosphorylation at specific sites by highly specific protein tyrosine kinases. These and other selected biochemical studies that contribute to the evolving view of in vitro substrate specificity of protein tyrosine kinases are reviewed.