The prognostic significance of p34cdc2 and cyclin D1 protein expression in prostate adenocarcinoma

BACKGROUND: Cyclin-dependent kinases (CDK) and cyclins constitute the subunits of the maturation-promoting factor that controls the process of cell division. High levels of these proteins have been reported in human malignancies of the stomach, colon, breast, and lung, and have been implicated in aberrant cell division and dysregulated tumor growth. METHODS: p34cdc2 CDK and cyclin D1 (D1) protein expression were evaluated in 140 radical prostatectomy specimens harboring adenocarcinoma (PAC), using the respective monoclonal antibodies on archival tissue sections. In each case, slides stained with hematoxylin and eosin were examined for evaluation of Gleason's grade and pathologic stage. The DNA content of the tumors was determined by the Feulgen method with the CAS200 Image Analyzer (Cell Analysis Systems, Lombard, IL). Nuclear immunoreactivity for the two proteins was semiquantitatively scored, and results were correlated with Gleason's grade, stage, ploidy, metastatic status, and disease recurrence after radical prostatectomy. RESULTS: p34cdc2 was expressed in 84 of 140 PACs (60%) and correlated with high Gleason's grade (P = 0.0001), advanced pathologic stage (P = 0.01), nondiploid DNA content (P = 0.0001), and metastases (P = 0.04). On multivariate analysis using the Cox proportional hazards model, p34cdc2 immunoreactivity (P = 0.0001) and high Gleason's grade (P = 0.01) each independently predicted disease recurrence. When tumors were of low Gleason's grade and lacked p34cdc2 expression, 4 of 39 PACs (10%) recurred, as compared with 18 of 47 (38%) that recurred when tumors were of high Gleason's grade and expressed p34cdc2 protein. D1 was positive in 31 of 140 PACs (22%) and showed a trend (P = 0.07) of high Gleason's grade, but it did not reach statistical significance with any of the prognostic variables. In the majority of PACs expressing both p34cdc2 and D1 proteins, the adjacent benign prostate acini showed focal, scattered nuclear positivity of the basal and secretory epithelial cells. CONCLUSIONS: p34cdc2 is expressed in a majority of PACs and correlates with high Gleason's grade, advanced pathologic stage, nondiploid DNA content, and metastases. On multivariate analyses high Gleason's grade and p34cdc2 immunoreactivity predict disease recurrence independently of the pathologic stage. Thus, p34cdc2 appears to play a critical role in the evolution, proliferation, and spread of PACs and may be of prognostic value when applied to initial prostate tissue samples taken by needle biopsy.     

Fetal supraventricular tachycardia complicated by hydrops fetalis: a role for direct fetal intramuscular therapy

Maternally administered digoxin for the treatment of fetal supraventricular tachycardia (SVT) complicated by hydrops fetalis may be ineffective secondary to poor transplacental drug transfer. We present our experience with eight pregnancies treated with transplacental therapy or combined maternal and direct fetal intramuscular therapy. Response to treatment following maternal intravenous administration (MIV) of digoxin or a combination of fetal intramuscular (FIM) digoxin and MIV is described for eight hydropic fetuses during nine successful pharmacologic conversions. The MIV digoxin was administered using standard loading and maintenance protocols. FIM was administered at a dose of 88 micrograms/kg q 12-24 hours, to a maximum of three injections in the fetal buttock. Time to onset of the first two hours of sinus rhythm (TO2 degrees), time to onset > 90% sinus rhythm (TO > 90%), and time to resolution of hydrops fetalis (HF) were noted. The mean heart rate was 257 +/- 36 beats/minute and the mean gestational age was 29 +/- 4.8 weeks. Fetal SVT was due to a reentrant mechanism in all cases. For the three fetuses that underwent successful cardioversion following MIV digoxin (all required additional maternal antiarrhythmic drugs), TO2 degrees was 145 +/- 114 hours, TO > 90% was 176 +/- 55 hours, and HF resolved in 41 +/- 37 days. Initial combined FIM and MIV therapy in four fetuses resulted in a TO2 degrees of 5.5 +/- 4 hours, TO > 90% of 22 +/- 14 hours, and resolution of HF in 25 +/- 21 days. For the two failed cardioversions with transplacental treatment alone (one fetus had recurrent SVT with hydrops after initial successful cardioversion with MIV), TO2 degrees was 203 +/- 180 hours and TO > 90% was 313 +/- 270 hours. Once FIM was begun in these fetuses, TO2 degrees was 17 +/- 7 hours and TO > 90% was 60 +/- 13 hours; HF resolved in 45 days in one fetus, whereas the other fetus never had resolution of hydrops despite 100 days of antiarrhythmic therapy. Direct fetal intramuscular injection of digoxin combined with transplacental therapy appears to shorten the time to initial conversion of SVT and to sustain sinus rhythm in the fetus with SVT complicated by hydrops fetalis.     

Reversal of ras-induced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin

The plasma-membrane association and transforming activity of the ras oncoprotein p21 are dependent upon posttranslational farnesylation. Farnesyl synthesis and p21 ras farnesylation are inhibited by hydroxymethylglutaryl-CoA reductase inhibitors such as lovastatin. In this study, we examined whether lovastatin could reverse the transformed phenotype of a v-Ha-ras-transformed rat liver epithelial cell line (WB-ras cells) and if changes were associated with the enhancement of gap-junctional intercellular communication (GJIC). WB-ras cells grow in soft agar, have reduced GJIC, and are highly tumorigenic. Membrane association of p21 ras in these cells was inhibited after in vitro treatment with lovastatin (0.1-0.5 microM) for 48 h. Concomitantly, the cells displayed a more normal morphology, decreased growth in soft agar, and enhanced GJIC. These changes were prevented by cotreatment with mevalonic acid. The morphology and GJIC of rat liver epithelial cells transformed with other oncogenes (src, neu, and raf/myc) were not affected by lovastatin. Intrahepatic WB-ras tumors were induced in male rats by intraportal-vein injection of WB-ras cells. The size and DNA labeling index of these tumors were decreased approximately 75% by administration of lovastatin (5 mg/kg orally twice daily for 2 wk). These results suggest that lovastatin reversed the transformed phenotype of WB-ras cells by inhibiting p21 ras plasma membrane association. Furthermore, the concomitant enhancement of GJIC in lovastatin-treated cells suggests a role for reduced GJIC in the expression of the transformed phenotype.     

Application of thrombin based fibrin glue and non-thrombin based batroxobin glue on intact human blood vessels: evidence for transmural thrombin activity

An alternative method of uniting small diameter vessels to obtain tissue union while limiting the thrombogenic effect of suture placement at a vessel anastomosis involves the use of a thrombin based fibrin glue as a surgical sealant. This investigation addresses whether the in vitro application of a thrombin based glue (TG), or batroxobin glue (BG), a non-thrombin based glue made with the snake venom enzyme batroxobin, alters intravascular platelet deposition (PD) or cleaves blood fibrinogen, as measured by fibrinopeptide A (FPA) production, when the respective glue is applied to the external surface of an intact human placental artery or an artery with an anastomosis. When TG was applied to the adventitial surface of an intact vessel or an anastomosis (n = 7) of control and experimental vessels, there was a significant increase in intraluminal platelet deposition, an effect not realized with BG (n = 12, intact vessel TG p = 0.01, BG p = 0.66, anastomosis TG p <0.01, BG p <0.01). Both TG and BG significantly increased FPA levels when human whole blood was perfused through both intact vessels or vessels containing an anastomosis when compared to control vessels (intact vessel TG and BG p <0.01, anastomosis TG and BG p <0.01). Labelled thrombin studies document the rapid passage of thrombin through an intact vessel wall or vessels with an anastomosis when TG was applied to the adventitial surface of the vessel. The data suggest that TG and BG are drug delivery systems for their respective enzymes that either pass through or transfer a message across not only a surgically created anastomosis, but also an intact vessel wall.     

Low de novo glutathione synthesis from circulating sulfur amino acids in the lens epithelium

Transport of circulating sulfur amino acids (SAA) into the lens epithelium and de novo glutathione (GSH) synthesis were studied in the perfused guinea-pig eye. Plasma-to-aqueous transfer of SAA was in their intact form (> or = 98%) and comparable with sucrose (an extracellular marker) within 30 min. The unidirectional transport rates (ml min-1 g-1) of 35S-labeled cystine, cystine and methionine into the epithelium were: 0.0057, 0.0003 and 0.0073 from plasma, and 1.41, 0.005 and 1.69 from aqueous, respectively. The unidirectional epithelial uptake was limited to 1 min for all three [35S]SAA, and the isotopic steady-state ratio was achieved between 1 and 30 min. Cortical uptake was time-dependent and progressive between 1 and 30 min, but undetectable within 1 min. The high performance liquid chromatography (HPLC) analysis of the epithelium revealed that following 1 min of unidirectional [35S]cysteine transport, 3% of the label was incorporated into GSH and > or = 95% was as cysteine. An average incorporation of [35S]cysteine into GSH within the 30 min period was 0.83% min-1 and 1%/min for the epithelium and cortex, respectively. Infusions of [35S]cystine and methionine failed to demonstrate incorporation into GSH. Maximal rates of de novo GSH epithelial synthesis were approximately 3 and 12 pmol g-1 from plasma and aqueous cysteine, respectively. A t1/2 of 5480 hr was estimated if epithelial GSH had to be replaced exclusively by synthesis from aqueous cysteine. Given the limited aqueous and epithelial cysteine pools, and low (from cysteine) or undetectable (from cystine and methionine) incorporation of the label into GSH, we conclude that de novo GSH synthesis from circulating and aqueous SAA can be only a minor source of the millimolar concentration of GSH in the epithelium.