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.     

p34cdc2 in invasive breast cancer: relationship to DNA content, Ki67 index and c-erbB-2 expression

AIMS: One-hundred and eighty-eight cases of human mammary carcinoma were examined immunohistochemically for their expression of Ki67, p34cdc2 and c-erbB-2. DNA image cytometry was performed to evaluate DNA ploidy, Auer type, S-phase fraction (SPF), 5c exceeding rate (5cER) and 2c deviation index (2cDI). METHODS AND RESULTS: One-hundred and sixty-eight cases were invasive ductal carcinomas, 20 were of invasive lobular type. Routinely assessed oestrogen and progesterone receptor scores were available. The results were analysed statistically in comparison to tumour type, histopathological grade, lymph node status, menopausal status, patient age and overall survival. Ki67 (P < 0.002) and c-erbB-2 (P < 0.0001) correlated well with overall survival (P < 0.0008) and grade (P < 0.038) but not with lymph node status and tumour type. p34cdc2 showed a trend towards a positive correlation with Ki67 (P < 0.058) and a significant negative correlation with receptor status (P < 0.008) but with none of the other parameters examined. CONCLUSIONS: No association between the DNA measured parameters (Auer type, SPF, 5cER and 2cDI) and survival was found. Our results suggest that c-erbB-2 and Ki67 are parameters which might, in combination with receptor status, help to define subgroups with different outcomes.     

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.     

Cisplatin-induced inhibition of p34cdc2 is abolished by 5-fluorouracil

This paper attempts to establish whether dissatisfaction with the artificial limb and/or body image relate to achieved mobility following lower limb amputation in established limb wearers. Patients attending limb fitting clinics (n = 107, 62% male, mean time from amputation 13.9 years; range 1-54) participated. The measures were a specially designed Attitude to Artificial Limbs Questionnaire, a Body Image Questionnaire adapted from an eating disorders instrument including reference to body shape, the Hospital Anxiety and Depression Scale and the Harold Wood Stanmore Mobility Scale. The rehabilitation physician rated prosthetic suitability on a Numerical Rating Scale. The results showed patients were moderately satisfied with their artificial limb, had little experience of body image disruption or distress and there was no overall relationship between these variables and mobility. However, those with a more negative body image were more anxious and in younger patients who sustained more traumatic than vascular amputations, the correlation between body image and mobility was significant, anxiety was higher and physician satisfaction with the prosthesis was lower. It is concluded that body image disruption, anxiety and depression are not common in established limb wearers except in young people with traumatic amputations.     

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.     

Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes

Increased expression of antioxidant enzymes and heat-shock proteins are key markers of oxidative stress. Such proteins are abnormally present within the neuropathological lesions of Alzheimer's disease (AD), suggesting that oxidative stress may play significant but yet undefined roles in this disorder. To gain further insight into the role of oxidative stress in AD, we studied the expression of CuZn superoxide dismutase (SOD) and hemoxygenase-1 (HO-1), two established markers of oxidative stress, in a transgenic mouse model of AD. Immunohistochemistry with anti-SOD and anti-HO-1 antibodies revealed a very pronounced increase of these proteins only in aged transgene-positive mice. Interestingly, the distribution of the oxidative burden was largely overlapping with dystrophic neuritic elements in the mice as highlighted with anti-ubiquitin antibodies. Because the most conspicuous alterations were identified around amyloid (Abeta) deposits, our results provide strong support for the hypothesis that Abeta is neurotoxic in vivo and that such toxicity is mediated by free radicals. To obtain additional experimental evidence for such an interpretation (ie, a cause-effect relationship between Abeta and oxidative neurotoxicity), PC12 cells were exposed to increasing concentrations of Abeta or to oxidative stress. In agreement with the in vivo findings, either treatment caused marked induction of SOD or HO-1 in a dose-dependent fashion. These results validate the transgenic approach for the study of oxidative stress in AD and for the evaluation of antioxidant therapies in vivo.     

Altered cell shape is linked to increased p34cdc2 gene expression in fibroblasts expressing a mutant E2F-1 transcription factor

Transhydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprising, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H)--or its analogues and NADP(H)--or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation(a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (< 5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with 'normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH-->AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.     

Induction of p34cdc2 in mouse parotid glands upon activation of beta1-adrenergic receptors

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites. These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene. To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil. Compatible plasmids carrying the different attB regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision. This experimental system was used to study the sequences required in attB for efficient site-specific recombination. A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies. By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.     

Cellular kinetic differences between Hodgkin''s and anaplastic large cell lymphomas: relation to the expression of p34cdc2 and cyclin B-1

Normal human subjects were tested for their ability to discriminate the orientation of a square plaque tilted in depth, using two different tasks: a grasping task and a perceptual matching task. Both tasks were given under separate monocular and binocular conditions. Accuracy of performance was measured by use of an opto-electronic motion analysis system, which computed the hand orientation (specifically, a line joining the tips of the thumb and index finger) as the hand either approached the target during grasping or was used to match the target. In all cases there was a very strong statistical coupling between hand orientation and target orientation, irrespective of viewing conditions. However, the matching data differed from the grasping data in showing a consistent curvature in the hand-target relationship, whereby the rate of change of hand orientation as a function of object orientation was smaller for oblique orientations than for those near the horizontal or vertical. The results are interpreted as reflecting the operation of two different mechanisms for analysing orientation in depth: a visuomotor system (assumed to be located primarily in the dorsal cortical visual stream) and a perceptual system (assumed to be located in the ventral stream). It may be that the requirements of visuomotor control dictate a primary need for absolute orientation coding, whereas those of perception dictate a need for more categorical coding.     

Abnormalities in the p34cdc2-related PITSLRE protein kinase gene complex (CDC2L) on chromosome band 1p36 in melanoma

Recently, several clusters of hepatitis A have been observed among hemophiliacs linked to factor VIII concentrates treated for virus inactivation solely with the solvent/detergent (S/D) method, a procedure that does not affect nonenveloped viruses such as the hepatitis A virus (HAV). A new outbreak of hepatitis A in six hemophiliacs treated with the same lot of a factor VIII preparation occurred recently in Germany. The objective of the study was to clarify whether these diseases were caused by the administration of the S/D-treated plasma product, rather than a community-acquired infection. Polymerase chain reactions designed to detect HAV nucleic acid have been carried out in the implicated factor VIII lots, in the corresponding plasma pools, and in serum samples of four out of six infected individuals. The nucleic acid sequences were determined in samples that resulted in positive amplification products. HAV sequences were found in one of the two plasma pools used for manufacture of the incriminated product, in the incriminated lot itself, and in all recipient sera tested so far, although the latter were collected up to 7 weeks after the onset of jaundice. The sequences obtained were completely identical, revealing a unique HAV strain of genotype IA. This study provides conclusive evidence that hepatitis A can be transmitted by factor VIII concentrates treated solely by the S/D procedure for virus inactivation. This inactivation method is not effective against nonenveloped viruses. Since a number of hepatitis A transmission episodes have been described with such preparations during the past 10 years, their continued use seems to be questionable unless additional virus removal or inactivation steps are introduced to prevent the transmission of nonenveloped viruses. Molecular approaches again proved to be reliable tools for elucidating the chain of virus transmission.     

Two functional soybean genes encoding p34cdc2 protein kinases are regulated by different plant developmental pathways

We have isolated two cDNA clones (cdc2-S5 and cdc2-S6) encoding p34cdc2 protein kinases, homologs of yeast cdc2/CDC28 genes, from a soybean nodule cDNA library. The two sequences share 90% sequence homology in the coding regions. The 5' and 3' noncoding regions are distinct from each other, however, indicating that at least two genes encode p34cdc2 protein kinases in soybean. Both sequences can rescue the cdc28 mutation in Saccharomyces cerevisiae but rescue it with different efficiency. Genomic Southern analysis showed the existence of two copies for each of these genes, which are not closely linked and are nonallelic. The relative expression level of the two soybean p34cdc2 genes varies in different tissues. Expression of cdc2-S5 is higher in roots and root nodules, whereas cdc2-S6 is more actively expressed in aerial tissues, indicating that regulation of these two p34cdc2 genes is coupled with plant developmental pathways. Expression of cdc2-S5 is, furthermore, enhanced after Rhizobium infection, whereas cdc2-S6 fails to respond, suggesting that cdc2-S5 plays a role in nodule initiation and organogenesis. This latter gene preferentially responds to auxin (alpha-naphthaleneacetic acid) treatment, indicating that phytohormones may be involved in the control of cell division mediated by Rhizobium infection. Thus, different p34cdc2 protein kinases may control cell division in different tissues in a multicellular organism and respond to different signals--e.g., phytohormones.     

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.     

Strain difference in the timing of meiosis resumption in mouse oocytes: involvement of a cytoplasmic factor(s) acting presumably upstream of the dephosphorylation of p34cdc2 kinase

A group of 39 rats underwent excision of the submandibular and sublingual glands and ligation of the parotid ducts through the midventral incision of the neck, while the control group (41 rats) underwent a sham operation. All rats were administered 4NQO in a final concentration of 0.001% in drinking water. At 7, 14, 22 and 28 weeks after administering 4NQO, both groups of rats were killed and their tongues dissected, inspected and then fixed in 10% buffered formalin for histopathological examination. Clinical examination during the first 14 weeks revealed that rats in both groups looked healthy and no differences in body weight were noticed. Afterwards, the average weight gain of the desalivated rats was lower than in the control group (P < 0.01). The number of macroscopic oral lesions increased with time in both groups. However, in the desalivated rats, the first identifiable lesions were seen as early as week 7, whereas in the control group macroscopic lesions were seen only after 22 weeks. Histological examination revealed more affected rats in the desalivated group in the first 14 weeks after administering the carcinogen; lesions showed more severe pathological changes including two cases with evidence of squamous cell carcinoma. The differences between the desalivated groups and control decreased after 22 weeks with almost no differences at the end of the experiment.     

Petunia p34cdc2 protein kinase activity in G2/M cells obtained with a reversible cell cycle inhibitor, mimosine

Protoplasts isolated from petunia leaf mesophyll are non-cycling cells mostly with 2C content. Cells regenerating from protoplast culture enter mitosis after 48 h. This experimental model is used to relate p34cdc2 kinase activity to cell cycle phase. Our results show that the histone H1 phosphorylation, and hence p34cdc2 kinase activity, peaks with G2+early M cell cycle phase. However, a trace kinase activity was already present when most cells were entering S phase. To obtain a maximum of cells in G1+S phases, the protoplast culture was treated with the rare amino acid, mimosine. Mimosine blocked plant cells derived from protoplast culture both at G1 and in early and mid S phase. Despite the increased G1+S level, p34cdc2 kinase activity did not increase. This suggests that the trace activity appearing when the majority of cells are entering S does not correspond to any putative p34cdc2 activation at G1/S transition but to the activation of the minor 4C population initially present in the leaf: the hypothesis remains that p34cdc2 kinase activity is solely related to G2+M phase in petunia.     

Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition

The G2-M transition of the cell cycle is triggered by the p34(cdc2)/cyclin B kinase. During the prophase/metaphase transition, the inactive, Thr-14/Tyr-15 phosphorylated form of p34(cdc2) (TP-YP) is modified to an active, Thr-14/Tyr-15 dephosphorylated form (T-Y) by the cdc25 dual-specificity phosphatase. Using highly synchronized starfish oocytes as a cellular model, we show that dephosphorylation in vivo and in vitro occurs in two steps: Thr-14 dephosphorylation precedes Tyr-15 dephosphorylation. The transient intermediate form (T-YP), which can be obtained in vitro by treatment of TP-YP by protein phosphatase 2A, displays low but significant kinase activity. These results raise the possibility that the intermediate form T-YP may be involved in the autocatalytic amplification of the p34(cdc2)/cyclin B complex through phosphorylation/activation of the cdc25 phosphatase and phosphorylation/inactivation of the wee1 kinase.     

Sustained accumulation of the mitotic cyclins and tyrosine-phosphorylated p34cdc2 in human G1-S-arrested cancer cells but not untransformed cells

Coupling mitosis to the completion of DNA replication in cycling embryonic extracts from Xenopus eggs appears to rely on blocking the activation of the tyrosine-phosphorylated p34cdc2/cyclin B, which continues to build up when S phase is inhibited by adding unreplicated DNA (Smythe, C., and Newport, J. W., Cell, 68: 787-797, 1992). We show here that a similar mechanism might be operative in human tumor-derived cells, which, during a thymidine-aphidicolin block, stop progressing through S phase and thereby fail to undergo mitosis. Under such conditions, indeed, cancer cells do continue to accumulate cyclin A, cyclin B1, and tyrosine-phosphorylated p34cdc2 to supranormal levels, a phenomenon that does not occur in untransformed, nonimmortalized human fibroblasts. Thus, in human cancer cells, the onset of active accumulation of cyclin A and cyclin B1 can be uncoupled from transit through the G1-S and S-G2 borders, respectively, and, as in simple embryonic cell cycles, the coupling of mitosis to the completion of S phase presumably relies, at least in part, on the prevention of premature activation of the tyrosine-phosphorylated p34cdc2/cyclin B1 complex.     

Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites

The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.     

Correlation of p34cdc2 cyclin-dependent kinase overexpression, CD44s downregulation, and HER-2/neu oncogene amplification with recurrence in prostatic adenocarcinomas

PURPOSE: To test whether p34cdc2 overexpression, CD44s downregulation, and HER-2/neu amplification correlate with disease recurrence after radical prostatectomy, and to evaluate a possible biologic association between p34cdc2 and HER-2/neu expression. MATERIALS AND METHODS: Immunohistochemical (IHC) detection of both p34cdc2 cyclin-dependent kinase (CDK) and CD44s expression and fluorescence in situ hybridization (FISH)-based analysis of HER-2/neu gene status were performed on formalin-fixed, paraffin-embedded sections of 106 prostatic adenocarcinomas (PACs). Findings were correlated with Gleason grade, pathologic stage, DNA ploidy, and postsurgical biochemical disease recurrence. RESULTS: CDK overexpression correlated with tumor grade (P = .001), DNA ploidy (P = .001), pathologic stage (P = .04), and disease recurrence (P = .01). CD44s downregulation correlated with grade (P = .03), ploidy (P = .01), and recurrence (P = .02). HER-2/neu amplification correlated with grade (P = .001), ploidy (P = .001), and recurrence (P = .01). On multivariate analysis, CDK overexpression independently predicted recurrence (P = .001) after prostatectomy. CDK expression correlated with HER-2/neu status with 32 of 65 (49%) tumors that overexpressed CDK and showed concomitant HER-2/neu amplification (P = .04). CONCLUSION: This study showed that p34cdc2, CD44s, and HER-2/neu are variably expressed or amplified in prostatic carcinoma and that such alteration may affect tumor behavior. In addition, CDK overexpression and HER-2/neu amplification may be biologically related.     

Caffeine release of radiation induced S and G2 phase arrest in V79 hamster cells: increase of histone messenger RNA levels and p34cdc2 activation

The serine/threonine protein kinase p34cdc2 activity in V79 hamster cells 4 h after treatment with 7-Gy X-rays is similar to that of unirradiated cells. Nevertheless, the irradiated cells are arrested in the S and G2 phases of the cell cycle. The mRNA concentrations of histones H1 and H4 are reduced by a factor of about 2 in irradiated cells compared to unirradiated cells, as opposed to the mRNAs of high-mobility group I(Y) and 17 proteins which appear unchanged. Both the p34cdc2 activity and the mRNA concentrations of the histones rise within 30 min after the release of the radiation induced cell cycle block by caffeine. During this time span the p34cdc2 activity increases about 4-fold and the histone mRNA levels recover approximately to those of an exponentially growing cell population. Regulatory pathways influenced in irradiated and in subsequently caffeine treated cells apparently interact with basic cell cycle control mechanisms.