Elevation of cyclic adenosine 3',5'-monophosphate potentiates activation of mitogen-activated protein kinase by growth factors in LNCaP prostate cancer cells

OBJECTIVE: Proinflammatory cytokines are involved in the pathogenesis of acute pancreatitis. The value of serum levels of tumor necrosis factor-alpha, interleukin-1-beta, interleukin-6, and interleukin-8 in predicting the outcome of acute pancreatitis was evaluated. METHODS: In 50 patients with acute pancreatitis, the serum concentrations of tumor necrosis factor-alpha, interleukin-1-beta, interleukin-6, interleukin-8, and C-reactive protein were determined on days 1, 2, 3, 4, and 7 after admission. Acute Physiology and Chronic Health Evaluation (APACHE II) scores were recorded on days 1, 2, and 3. RESULTS: Serum concentrations of interleukin-1-beta, interleukin-6, interleukin-8, and C-reactive protein on days 1-7 were significantly higher in patients with severe pancreatitis than in patients with mild pancreatitis. Patients with severe attacks had significantly elevated serum tumor necrosis factor-alpha concentrations on days 1-3 compared with those with mild attacks, but not on days 4 and 7. The median peak value of tumor necrosis factor-alpha, interleukin-1-beta, interleukin-6, and interleukin-8 was reached on day 1, in contrast to the median peak of C-reactive protein, which was reached on day 2. Using cutoff levels of 12 pg/ml for tumor necrosis factor-alpha, 1 pg/ml for interleukin-1-beta, 400 pg/ml for interleukin-6, 100 pg/ml for interleukin-8, 12 mg/dl for C-reactive protein, and 10 for the Acute Physiology and Chronic Health Evaluation (APACHE II) score, the accuracy rates for detecting severe pancreatitis were 72%, 82%, 88%, 74%, 80%, and 72%, respectively, on day 1 and 78%, 74%, 80%, 76%, 80%, and 78%, respectively, on day 2. CONCLUSION: Among the proinflammatory cytokines, interleukin-6 is the most useful parameter for early prediction of the prognosis of acute pancreatitis.     

Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein

Escherichia coli cAMP receptor protein (CRP) regulates the expression of a large number of catabolite-sensitive genes. The mechanism of CRP regulation most likely involves communication between subunits and domains. A specific message, such as the activation of CRP, may be manifested as a change in the interactions between these structural entities. Hence, the elucidation of the regulatory mechanism would require a quantitative evaluation of the energetics involved in these interactions. Thus, a study was initiated to define the conditions for reversible denaturation of CRP and to quantitatively assess the energetics involved in the intra- and intersubunit interactions in CRP. The denaturation of CRP was induced by guanidine hydrochloride. The equilibrium unfolding reaction of CRP was monitored by three spectroscopic techniques, namely, fluorescence intensity, fluorescence anisotropy, and circular dichroism. The spectroscopic data implied that CRP unfolds in a single cooperative transition. Sedimentation equilibrium data showed that CRP is dissociated into its monomeric state in high concentrations of denaturant. Unfolding of CRP is completely reversible, as indicated by fluorescence and circular dichroism measurements, and sedimentation data indicated that a dimeric structure of CRP was recovered. The functional and other structural properties of renatured and native CRP have also been examined. Quantitatively identical results were obtained. Results from additional studies as a function of protein concentration and from computer simulation demonstrated that the denaturation of CRP induced by guanidine hydrochloride proceeds according to the following pathway: (CRP2)Native<-->2(CRP)Native<-->2(CRP)Denatured. The delta G values for dissociation (delta Gd) and unfolding (delta G(u)) in the absence of guanidine hydrochloride were determined by linear extrapolation, yielding values of 12.0 +/- 0.6 and 7.2 +/- 0.1 kcal/mol, respectively. To examine the effect of the DNA binding domain on the stability of the cAMP binding domain, two proteolytically resistant cAMP binding cores were prepared from CRP in the presence of cAMP by subtilisin and chymotrypsin digestion, yielding S-CRP and CH-CRP, respectively. Results from an equilibrium denaturation study indicated that the denaturation of both CH-CRP and S-CRP is also completely reversible. Both S-CRP and CH-CRP exist as stable dimers with similar delta Gd values of 10.1 +/- 0.4 and 9.5 +/- 0.4 kcal/mol, respectively. Results from this study in conjunction with crystallographic data [McKay, D. B., Weber, I. T., & Stietz, T. A. (1982) J. Biol. Chem. 257, 9518-9524] indicate that the DNA binding domain and the C-helix are not the only structural elements that are responsible for subunit dimerization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic adenosine 3',5'-monophosphate-binding proteins in human ovarian cancer: correlations with clinicopathological features

The regulatory subunits of protein kinase A, or cyclic AMP-binding proteins, were measured in a series of 107 human ovarian tumors (89 malignant, 7 borderline, and 11 benign tumors) and related to tumor clinicopathological features and patient survival. Total cyclic AMP-binding protein levels were not significantly different between malignant tumors and either borderline or benign tumors. However, serous tumors showed significantly higher levels of total cyclic AMP-binding proteins than other malignant tumors (P = 0.007). Poorly differentiated tumors also possessed significantly higher levels of binding proteins as compared with well/moderately differentiated tumors (P < 0.01). Retrospective analysis of follow-up data also revealed a significant trend for patients with high tumor cyclic AMP-binding proteins to have poorer survival (P = 0.03). Individual binding proteins were identified by photoaffinity labeling, and the RI (Mr 48,000) protein was expressed as a percentage of total cyclic AMP-binding proteins detected. The percentage of the RI protein was not significantly different among malignant, borderline, or benign pathologies and was not associated with tumor stage, differentiation, or debulk status. The percentage of RI was significantly increased in serous tumors compared to other common epithelial malignancies (P = 0.01). In malignant tumors there was a significant positive correlation between the percentage of the RI protein and total cyclic AMP-binding proteins (P = 0.01). These data indicate that high tumor levels of cyclic AMP-binding proteins are associated with serous histology, poor differentiation, and poor patient survival.     

Calcitonin stimulates growth of human prostate cancer cells through receptor-mediated increase in cyclic adenosine 3',5'-monophosphates and cytoplasmic Ca2+ transients

Our recent study has shown that a calcitonin (CT)-like immunoreactive substance(s) is secreted by cultured prostate cells, and secretion of this material is significantly higher in malignant than in benign prostate cells. To test the hypothesis that prostatic CT may serve as a paracrine/neuroendocrine factor, the present study investigated for the presence of CT receptors in the prostate gland. Signal transduction mechanisms activated by CT were examined, and the study also tested its effects on prostate cell proliferation, as assessed by [3H]thymidine incorporation. The results show that high affinity binding sites for [125I]salmon CT were present in plasma membrane fractions of human prostate tissue specimens and the prostate cancer LnCaP cell line. The maximal binding for CT receptors was 564 +/- 163 fmol/mg protein, and the apparent dissociation constant (Kd) was 2.89 +/- 0.58 nM. CT induced a dose-dependent increase in cAMP generation in LnCaP cells. The effect of CT on cytoplasmic Ca2+ transients of LnCaP cells was examined by videofluoromicroscopy. CT (100 nM) induced a rapid and sharp increase in cytoplasmic Ca2+ concentrations in LnCaP cells. The CT-induced increase in cytoplasmic Ca2+ transients appeared to be biphasic (spike and plateau), and this increase was 4- to 10-fold during the initial phase. The profile of this response is characteristic of the activated Ca2+/phospholipid second messenger system. CT also caused a dose-dependent increase in [3H]thymidine incorporation by LnCaP cells. These results suggest that a locally secreted CT-like peptide(s) induces mitogenic responses in prostate cancer cells. This action seems to be mediated through activation of signaling mechanisms, leading to the accumulation of two different second messengers, cAMP and calcium. Activation of dual second messenger systems by CT receptors suggests that the peptide hormone may play an important role in rapidly growing cell populations during the process of tumor formation.     

Evidence for a role for the cyclic adenosine 3',5'-monophosphate/protein kinase-A pathway in regulation of the gonadotropin subunit messenger ribonucleic acids

cAMP regulation of gonadotropin secretion and subunit mRNA levels was studied in pituitary cells perifused with pulses of GnRH. Pituitary cells from 7-week-old male rats castrated at 5 weeks of age were stimulated hourly for 9-24 h with 1-min pulses of GnRH, the adenylate cyclase activator forskolin, the cell-permeable cAMP analog 8-bromo-cAMP (8Br-cAMP), or control medium. Cells were also treated with the nonsteroidal antiinflammatory drug flufenamic acid, which reduces pituitary cAMP levels. During perifusion, the effluent was collected in 10-min fractions for FSH and LH assay. At the completion of perifusion, total RNA was extracted, and gonadotropin subunit mRNA levels were quantitated by Northern analysis. Continuous administration of flufenamic acid gradually reduced the amplitude of GnRH-stimulated FSH and LH pulses to nadir values of 40 +/- 4.7% and 62 +/- 12% of the control value, respectively. Flufenamic acid decreased (P < 0.05) FSH beta and alpha-subunit mRNA levels and blocked the effect of GnRH to lengthen LH beta mRNA. Pulses of forskolin or 8Br-cAMP released LH and FSH, and continuous forskolin or 8Br-cAMP potentiated the gonadotropin stimulatory effect of GnRH. Forskolin or 8Br-cAMP increased (P < 0.05) FSH beta mRNA and alpha-subunit mRNA levels when administered in pulses, but not when administered continuously, and lengthened LH beta mRNA. The Nal-Glu GnRH antagonist blocked the effects of GnRH pulses, but not the effects of 8Br-cAMP or forskolin. In conclusion, lowering intracellular cAMP levels with flufenamic acid attenuated GnRH-stimulated gonadotropin secretion, decreased alpha-subunit and FSH beta mRNA levels, and blocked the effect of GnRH to lengthen LH beta mRNA, whereas 8Br-cAMP or forskolin produced the opposite effect. These data extend previous results which suggested that cAMP modulates gonadotropin secretion and indicate that the cAMP/A-kinase pathway regulates each of the gonadotropin subunit mRNAs.     

Induction of type 2 deiodinase activity by cyclic guanosine 3',5'-monophosphate in cultured rat glial cells

We investigated the effects of cyclic guanosine 3',5'-monophosphate (cGMP) on type 2 iodothyronine deiodinase (D2) in cultured rat glial cells. Rat glial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum. When cells were cultured in the presence of 8-bromo cGMP (8-Br cGMP), an analogue of cGMP, D2 activity was increased in a time- and concentration-dependent manner. Lineweaver-Burk plots revealed that the stimulation of D2 activity by 8-Br cGMP (10(-3) M) was associated with fivefold increase in maximum velocity but without a significant change in Michaelis-Menten constant, suggesting that cGMP increases D2 activity via new enzyme synthesis. Both atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) are well known to increase the intracellular cGMP level via their guanylate cyclase-linked receptors in rat glial cells. In the present study, ANP (10(-6) M) and CNP (10(-6) M) significantly increased the D2 activity in rat glial cells (1.9-fold [ANP] or 2.3-fold [CNP] compared with control activity, respectively). Northern blot analysis demonstrated that D2 mRNA level increased in the presence of 8-Br cGMP (10(-3) M), and reached a plateau (six-fold) after 4 hours of incubation. The increment of D2 mRNA level by 8-Br cGMP was comparable with the increase of the D2 activity by this agent. Our data suggest that cGMP induces rat D2 activity, at least in part, at the pretranslational level, and that ANP and CNP increase D2 activity most likely via their guanylate cyclase-linked receptors in rat glial cells.