Structure-activity relationship studies of flavopiridol analogues.

Cyclin dependent kinases (CDKs) along with the complementary cyclins form key regulatory checkpoint controls on the cell cycle. Flavopiridol is a synthetic flavone that shows potent and selective cyclin-dependent kinase inhibitory activity. In this paper, we report modifications of the 3-hydroxy-1-methylpiperidinyl (D ring) of flavopiridol and their effect on CDK inhibitory activity.

Mechanism of Cdk2/Cyclin E inhibition by p27 and p27 phosphorylation.

The biochemical interactions between the Cdk2/Cyclin E kinase and its inhibitor p27, were investigated using purified, recombinant p27 and CAK-phosphorylated Cdk2/Cyclin E. From kcat/Km determinations using either histone H1 or pRb as substrates, we found that Cdk2/Cyclin E has 60-fold higher specificity for pRb than for histone H1. The IC50 value of p27 increased with increasing Cdk2/Cyclin E concentrations while it remained constant at various ATP and histone H1 concentrations, suggesting that p27 acts as a tight binding inhibitor of Cdk2/Cyclin E. We also found that p27 could be phosphorylated by Cdk2/Cyclin E only at high enzyme concentrations, and that p27 forms a stable interaction with Cdk2/Cyclin E regardless of its phosphorylation state. Our results further indicate that the Cdk2/Cyclin E/p27 ternary complex is kinetically inactive as an enzyme; instead it serves as a substrate for Cdk2/Cyclin E. These results suggest that if phosphorylation of p27 by Cdk2/Cyclin E is involved in its ubiquitin-dependent degradation, as previously suggested, then the target for such event is the phosphorylated p27 bound to Cdk2/Cyclin E and not free p27.

Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells.

Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G1 or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cyclin D1 and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigate the G1 arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (Kiapp, 65 nM), competitive with respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavonolpiridol) had an approximately 3-fold increased kinase activity compared with untreated cells. Cyclin D and CDK4 levels were not different at 3 hr, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphorylation state of pRb was shifted from hypercoincident to hypocoincident with the development of G1 arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G1 and G2 by Flavopiridol. Flavopiridol inhibited the in vitro kinase activity of CDK2 using an immune complex kinase assay (IC50, 100 nM at 400 microM ATP). Immunoprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cells exposed to Flavopiridol (300 nM) for increasing time showed an initial increased activity (approximately 1.5-fold at 3 h) compared with untreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cyclin E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol. Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol can therefore account for the G1 arrest observed after exposure to Flavopiridol.

Biochemical and mutagenic analysis of the melanoma tumor suppressor gene product/p16.

P16 was originally discovered by its ability to interact with CDK4 and to specifically inhibit the catalytic activity of the CDK4/D1 kinase. Increased attention has focused on the p16 gene because of its location on chromosome 9p21, a region involved in chromosomal rearrangements in a large number of tumor types. The p16 gene is also mutated in a large number of tumor cell lines and primary tumor cells. Furthermore, linkage analysis studies suggest that the p16 gene is involved in familial melanoma susceptibility. Due to the oncogenic potential of mutations in this tumor suppressor, it is important to identify and characterize those mutations which alter p16 activity. We have performed a systematic analysis of melanoma associated p16 mutants and of mutants generated in charge to Ala mutagenesis. Using microtiter plate assays to measure both p16-cdk4 binding and cdk4/D1 kinase activity, we show here that the melanoma associated mutants are defective, as are some of the Ala mutants. These results support the idea that p16 mutation, via its deregulation of the cdk4/D1 pathway, is of biological significance in the development of melanoma. Furthermore, we have defined a region within the p16 molecule in which changes are likely to result in a defective protein.

[A method for ascertaining of the closing volume with argon for early detection of obstruction of the small airways (author's transl)]. / Closing Volume-Bestimmungsmethode mittels Argon zum frühzeitigen Nachweis der Obstruktion der kleinen Bronchien.

Closing volume can be ascertained with Argon by the aid of the Quadrupol-mass-spectrometer produced in Hungary. The method is simple, sensible and applicable in the routine laboratorium for pulmonary function testing. It will show the obstruction of the small airways and the disturbance of gas distribution in the lower pulmonary parts already at a point of time when the usual spirometric methods are still inconspicuous.

[CO2-production of circulating lymphocytes in the presence of a sarcoidosis extract in patients with sarcoidosis (author's transl)]. / Steigerung der Kohlendioxydproduktion zirkulierender Lymphozyten bei Sarkoidosepatienten durch Zugabe eines Sarkoidose-Extraktes.

The CO2-production of circulating lymphocytes of patients with sarcoidosis and other diseases was investigated in presence of an sarcoidosis extract and without it. In 32 of 36 patients with sarcoidosis an elevated CO2-production of the lymphocytes was observed in presence of a sarcoid extract, whereas it happened only in 3 of 26 cases of a control group.

Preoperative hot conization of the cervix: a possible method to reduce postoperative febrile morbidity following vaginal hysterectomy.

Laboratory results indicate that the endocervix may be a source of bacterial contamination when vaginal hysterectomy is performed. In a series of 160 consecutive vaginal hysterectomies in premenopausal women, hot conization of the cervix was performed prior to the scrub with an iodophore. No preoperative antibiotics were used in this series. The postoperative febrile morbidity rate was 4.3 per cent and the average stay was 4.5 days. These results are compared with those of three other groups: (1) patients who received a three-dose parenteral prophylactic antibiotic course with the first dose two hours prior to surgery had a febrile morbidity rate of 8.6 per cent. (2) In patients who had prophylactic antibiotics for five days with the first dose given intraoperatively, the febrile morbidity rate was 10.1 per cent. (3) The febrile morbidity rate in the group with no antibiotic prophylaxis or hot conization was 49.1 per cent. Laboratory and clinical data suggest that preoperative conization may be effective in the reduction of postoperative febrile morbidity.