Reactivation of the mitosis-promoting factor in postmitotic cardiomyocytes

Cardiomyocytes cease to divide shortly after birth and an irreversible cell cycle arrest is evident accompanied by the downregulation of cyclin-dependent kinase activities. To get a better understanding of the cardiac cell cycle and its regulation, the effect of functional recovery of the mitosis-promoting factor (MPF) consisting of cyclin B1 and the cyclin-dependent kinase Cdc2 was assessed in primary cultures of postmitotic ventricular adult rat cardiomyocytes ( ARC). Gene transfer into ARC was achieved using the adenovirus-enhanced transferrinfection system that was characterized by the absence of cytotoxic events. Simultaneous ectopic expression of wild-type versions of cyclin B1 and Cdc2 was sufficient to induce MPF activity. Reestablished MPF resulted in a mitotic phenotype, marked by an abnormal condensation of the nuclei, histone H3 phosphorylation and variable degree of decay of the contractile apparatus. Although a complete cell division was not observed, the results provided conclusive evidence that cell cycle-related events in postmitotic cardiomyocytes could be triggered by genetic intervention downstream of the G1/S checkpoint. This will be of importance to design novel strategies to overcome the proliferation arrest in adult cardiomyocytes

Cryoultramicrotomy and Immunocytochemistry in the Analysis of Muscle Fine Structure

Cryoultramicrotomy, which avoids the use of harsh fixation procedures, deleterious dehydration and plastic embedding can be combined with immunocytochemis try to determine the ultra-structural localization of cellular proteins. Our attempts to use the cryosectioning technique in combination with immunolabelling to bridge the gap between light and electron microscopic analysis of muscle morphology have enabled us to obtain new information on fibre typing at the ultrastructural level. Furthermore, we have obtained a marked improvement in the resolution of myofibrillar structures by using semithin cryosections for fluorescence microscopy. Data are also presented on correlated light and electron microscope immunocytochemistry of myocardial intermediate filaments confirming the presence of longitudinally oriented intermediate filaments of desmin in the region of the intercalated discs of mammalian cardiac myocytes, whereas elsewhere in the myocyte the bulk of intermediate filaments of desmin is concentrated in the intermyofibrillar space at the level of the Z disc

Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients

BACKGROUND: Urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) play essential roles in tumor invasion and metastasis. High levels of both uPA and PAI-1 are associated with poor prognosis in breast cancer patients. To confirm the prognostic value of uPA and PAI-1 in primary breast cancer, we reanalyzed individual patient data provided by members of the European Organization for Research and Treatment of Cancer-Receptor and Biomarker Group (EORTC-RBG). METHODS: The study included 18 datasets involving 8377 breast cancer patients. During follow-up (median 79 months), 35% of the patients relapsed and 27% died. Levels of uPA and PAI-1 in tumor tissue extracts were determined by different immunoassays; values were ranked within each dataset and divided by the number of patients in that dataset to produce fractional ranks that could be compared directly across datasets. Associations of ranks of uPA and PAI-1 levels with relapse-free survival (RFS) and overall survival (OS) were analyzed by Cox multivariable regression analysis stratified by dataset, including the following traditional prognostic variables: age, menopausal status, lymph node status, tumor size, histologic grade, and steroid hormone-receptor status. All P values were two-sided. RESULTS: Apart from lymph node status, high levels of uPA and PAI-1 were the strongest predictors of both poor RFS and poor OS in the analyses of all patients. Moreover, in both lymph node-positive and lymph node-negative patients, higher uPA and PAI-1 values were independently associated with poor RFS and poor OS. For (untreated) lymph node-negative patients in particular, uPA and PAI-1 included together showed strong prognostic ability (all P<.001). CONCLUSIONS: This pooled analysis of the EORTC-RBG datasets confirmed the strong and independent prognostic value of uPA and PAI-1 in primary breast cancer. For patients with lymph node-negative breast cancer, uPA and PAI-1 measurements in primary tumors may be especially useful for designing individualized treatment strategies

Enhanced NFκB and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer

BACKGROUND: Signaling pathways that converge on two different transcription factor complexes, NFκB and AP-1, have been identified in estrogen receptor (ER)-positive breast cancers resistant to the antiestrogen, tamoxifen. METHODS: Two cell line models of tamoxifen-resistant ER-positive breast cancer, MCF7/HER2 and BT474, showing increased AP-1 and NFκB DNA-binding and transcriptional activities, were studied to compare tamoxifen effects on NFκB and AP-1 regulated reporter genes relative to tamoxifen-sensitive MCF7 cells. The model cell lines were treated with the IKK inhibitor parthenolide (PA) or the proteasome inhibitor bortezomib (PS341), alone and in combination with tamoxifen. Expression microarray data available from 54 UCSF node-negative ER-positive breast cancer cases with known clinical outcome were used to search for potential genes signifying upregulated NFκB and AP-1 transcriptional activity in association with tamoxifen resistance. The association of these genes with patient outcome was further evaluated using node-negative ER-positive breast cancer cases identified from three other published data sets (Rotterdam, n = 209; Amsterdam, n = 68; Basel, n = 108), each having different patient age and adjuvant tamoxifen treatment characteristics. RESULTS: Doses of parthenolide and bortezomib capable of sensitizing the two endocrine resistant breast cancer models to tamoxifen were capable of suppressing NFκB and AP-1 regulated gene expression in combination with tamoxifen and also increased ER recruitment of the transcriptional co-repressor, NCoR. Transcript profiles from the UCSF breast cancer cases revealed three NFκB and AP-1 upregulated genes – cyclin D1, uPA and VEGF – capable of dichotomizing node-negative ER-positive cases into early and late relapsing subsets despite adjuvant tamoxfien therapy and most prognostic for younger age cases. Across the four independent sets of node-negative ER-positive breast cancer cases (UCSF, Rotterdam, Amsterdam, Basel), high expression of all three NFκB and AP-1 upregulated genes was associated with earliest metastatic relapse. CONCLUSION: Altogether, these findings implicate increased NFκB and AP-1 transcriptional responses with tamoxifen resistant breast cancer and early metastatic relapse, especially in younger patients. These findings also suggest that agents capable of preventing NFκB and AP-1 gene activation may prove useful in restoring the endocrine responsiveness of such high-risk ER-positive breast cancers