[uPA/PAI-1 and EPClin®: Comparison of their impact on the management of intermediate-prognosis breast cancers]. / uPA/PAI-1 et EPClin® : comparaison de leur impact sur la prise en charge des cancers du sein de pronostic intermédiaire.

OBJECTIVE: The uPA/PAI-1 assay and the EPClin® test are useful tools that add to clinico-anatomical characteristics to determine the indication of adjuvant chemotherapy in case of intermediate-prognosis invasive breast cancer. The principal purpose of our study was to analyze the concordance of uPA/PAI-1 and EPClin® in classification of patients into two groups: low and high risk of relapse. METHODS: We prospectively included 63 patients treated for intermediate-prognosis invasive breast cancer. All of these patients received a uPA/PAI-1 assay and an EPClin® test. RESULTS: The uPA/PAI-1 assay and EPClin® test were consistent for 56.2% and inconsistent for 43.8%. In the event of a discrepancy, the treatment decision was based in 95.2% of patients on the EPClin® test result. In total, 38 patients were selected for adjuvant chemotherapy after achievement of the two tests. The mean time to report results after surgery was 9 days for the uPA/PAI-1 assay and 35 days for the EPClin® test. No cases of recurrence or death were found, with an average follow-up of 32 months. CONCLUSION: The EPClin® test resulted in more chemotherapy prescriptions than indicated by uPA/PAI-1. However, we can't conclude to the superiority of one of these two tests, survival data and the effectiveness of our study being insufficient. In general, studies comparing different signatures useful to the therapeutic decision of intermediate prognosis breast cancers should be encouraged.

Functional characterization of recombinant snake venom rhodocytin: rhodocytin mutant blocks CLEC-2/podoplanin-dependent platelet aggregation and lung metastasis.

Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and ß-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. SUMMARY: Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and ß-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTßWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTßWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTßK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.

C-type lectin-like receptor 2 promotes hematogenous tumor metastasis and prothrombotic state in tumor-bearing mice.

Essentials The role of C-type lectin-like receptor-2 (CLEC-2) in cancer progression is unclear. CLEC-2-depleted mouse model is generated by using a rat anti-mouse CLEC-2 monoclonal antibody. CLEC-2 depletion inhibits hematogenous tumor metastasis of podoplanin-expressing B16F10 cells. CLEC-2 depletion prolongs cancer survival by suppressing thrombosis and inflammation. SUMMARY: Background C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2-podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.

Physiologic and pathophysiologic roles of interaction between C-type lectin-like receptor 2 and podoplanin: partners from in utero to adulthood.

A platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2), has been identified as a receptor for a platelet-activating snake venom, rhodocytin. CLEC-2 protein is highly expressed in platelets/megakaryocytes, and at lower levels in liver Kupffer cells. Recently, podoplanin has been revealed as an endogenous ligand for CLEC-2. Podoplanin is expressed in certain types of tumor cells, fibroblastic reticular cells (FRCs) in lymph nodes, kidney podocytes, and lymphatic endothelial cells, but not in vascular endothelial cells. CLEC-2 in platelets cannot have access to podoplanin under normal conditions, but they interact with each other under pathologic conditions or during developmental stages, and play various pathophysiologic roles. CLEC-2 facilitates hematogenous metastasis of podoplanin-expressing tumors. During development, the interaction between CLEC-2 and podoplanin in lymphatic endothelial cells or neuroepithelial cells facilitates blood-lymphatic vessel separation and cerebrovascular patterning and integrity, respectively. In adulthood, platelet CLEC-2 binding to FRCs is crucial for maintenance of the integrity of high endothelial venules in lymph nodes. Podoplanin-expressing FRC-like cells have recently been identified in the bone marrow, and facilitate megakaryocyte proliferation and proplatelet formation by binding to megakaryocyte CLEC-2. Podoplanin is inducibly expressed in liver monocytes and keratinocytes during Salmonella infection and wound healing, and regulates thrombus formation in the liver and controlled wound healing, respectively. By binding to unknown ligands, platelet CLEC-2 regulates the maintenance of vascular integrity during inflammation, thrombus stability under flow, and maintenance of quiescence of hematopoietic stem cells. Podoplanin is expressed in various cells, and additional roles of the CLEC-2-podoplanin interaction will be revealed in the future.

p63(TP63) elicits strong trans-activation of the MFG-E8/lactadherin/BA46 gene through interactions between the TA and DeltaN isoforms.

We report here that human MFGE8 encoding milk fat globule-EGF factor 8 protein (MFG-E8), also termed 46 kDa breast epithelial antigen and lactadherin, is transcriptionally activated by p63, or TP63, a p53 (TP53) family protein frequently overexpressed in head-and-neck squamous cell carcinomas, mammary carcinomas and so on. Despite that human MFG-E8 was originally identified as a breast cancer marker, and has recently been reported to provide peptides for cancer immunotherapy, its transcriptional control remains an open question. Observations in immunohistochemical analyses, a tetracycline-induced p63 expression system and keratinocyte cultures suggested a physiological link between p63 and MFGE8. By reporter assays with immediately upstream regions of MFGE8, we determined that the trans-activator (TA) isoforms of p63 activate MFGE8 transcription though a p53/p63 motif at -370, which was confirmed by a chromatin immunoprecipitation experiment. Upon siRNA-mediated p63 silencing in a squamous cell carcinoma line, MFG-E8 production decreased to diminish Saos-2 cell adhesion. Interestingly, the DeltaN-p63 isoform lacking the TA domain enhanced the MFGE8-activating function of TA-p63, if DeltaN-p63 was dominant over TA-p63 as typically observed in undifferentiated keratinocytes and squamous cell carcinomas, implying a self-regulatory mechanism of p63 by the TA:DeltaN association. MFG-E8 may provide a novel pathway of epithelial-nonepithelial cell interactions inducible by p63, probably in pathological processes.

The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth.

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

Laboratory exposure to 17beta-estradiol fails to induce vitellogenin and estrogen receptor gene expression in the marine invertebrate Mytilus edulis.

To investigate the effect of estrogenic compounds on the marine mussel Mytilus edulis, an assay was developed to measure the expression of two vertebrate estrogen responsive genes-estrogen receptor (ER) and vitellogenin (VTG) genes. Expression was measured in M. edulis gonads following a 10-day exposure to 200 ng/l 17beta-estradiol (estradiol). The concentrations of esterified estradiol in mussel tissue increased 15-fold in a time-dependent manner-confirming uptake of the compound by the mussels, however there was no significant increase of free estradiol in mussel tissues during the exposure period. The ER and VTG mRNA levels in the gonads of both sexes were measured at days 1-3, 5, and 10 in control and exposed mussels. However, no significant change in the expression of either the ER or VTG genes was recorded at any of the sampled time points. The results suggest that either a regulatory mechanism exists in a mussel that is able to maintain constant levels of free estradiol by converting the excess estradiol into esterified products which may have reduced affinity for the estrogen receptor, or alternatively, that the ER and VTG genes are unresponsive to estrogens in these organisms. The significance of these findings in terms of the utility of ER and VTG as biomarkers of endocrine disruption in bivalve species is discussed.

Regulatory domain of protein stability of human P51/TAP63, a P53 homologue.

The amino terminal of human P51/TAp63, a P53 homologue, possesses a transactivation domain involved in the activation of its target genes by binding to DNA elements responsive to the p53 protein family. Using a series of amino terminal deletions, the transactivation domain was mapped between amino acid residues 50 to 69. This domain also regulates protein stability in a proteasome-dependent manner, and Ser51 and Ser68 were found to be essential for this stability. Our results suggest that P51 activity is greatly affected by protein stability.

Ischemic preconditioning prevents I/R-induced alterations in SR calcium-calmodulin protein kinase II.

Although Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) is known to modulate the function of cardiac sarcoplasmic reticulum (SR) under physiological conditions, the status of SR CaMK II in ischemic preconditioning (IP) of the heart is not known. IP was induced by subjecting the isolated perfused rat hearts to three cycles of brief ischemia-reperfusion (I/R; 5 min ischemia and 5 min reperfusion), whereas the control hearts were perfused for 30 min with oxygenated medium. Sustained I/R in control and IP groups was induced by 30 min of global ischemia followed by 30 min of reperfusion. The left ventricular developed pressure, rate of the left ventricular pressure, as well as SR Ca(2+)-uptake activity and SR Ca(2+)-pump ATPase activity were depressed in the control I/R hearts; these changes were prevented upon subjecting the hearts to IP. The beneficial effects of IP on the I/R-induced changes in contractile activity and SR Ca(2+) pump were lost upon treating the hearts with KN-93, a specific CaMK II inhibitor. IP also prevented the I/R-induced depression in Ca(2+)/calmodulin-dependent SR Ca(2+)-uptake activity and the I/R-induced decrease in the SR CaMK II activity; these effects of IP were blocked by KN-93. The results indicate that IP may prevent the I/R-induced alterations in SR Ca(2+) handling abilities by preserving the SR CaMK II activity, and it is suggested that CaMK II may play a role in mediating the beneficial effects of IP on heart function.

R-Ras3, a brain-specific Ras-related protein, activates Akt and promotes cell survival in PC12 cells.

The GTP-binding protein, R-Ras3/M-Ras, is a novel member of the Ras subfamily of GTPases which shows highest sequence similarity to the TC21 gene. R-Ras3 is highly expressed in both human and mouse brain and ectopic expression of a constitutively active mutant of R-Ras3 induces cellular transformation in NIH3T3 cells. To gain further insight into the normal cellular function of R-Ras3, we examined the ability of R-Ras3 in activating several known intracellular signaling cascades. We observed that R-Ras3 is a relatively weak activator of the mitogen-activated protein kinase/extracellular-signal-regulated kinases (MAPK/ERKs) when compared to the H-Ras oncogene. On the contrary, both R-Ras3 and H-Ras activated the Jun N-terminal kinase (JNK) to a similar extent. Under similar experimental conditions, R-Ras3 significantly stimulated one of the phosphatidylinositol 3-kinase (PI3-K) downstream substrates, Akt/PKB/RAC (Akt), which has been extensively implicated in mediating cell survival signaling. The activation of Akt by R-Ras3 was most likely to be PI3-K-dependent since this biochemical event was blocked by the pharmacological inhibitors, Wortmannin and LY294002, as well as by a dominant negative mutant of PI3-K. More importantly, R-Ras3 affinity-precipitated PI3-K from cell extracts in a GTP-dependent manner, and associated lipid kinase activity was readily detectable in R-Ras3 immune complexes. The biological significance of R-Ras3 in inducing Akt kinase activity is evidenced by the ability of an activated R-Ras3 to confer cell survival in the rat pheochromocytoma cell line, PC12. As expected, this biological activity of R-Ras3 was also abrogated by the addition of LY294002. Thus, R-Ras3 represents a novel G-protein which may play a role in cell survival of neural-derived cells.

Mechanisms of ischemic preconditioning effects on Ca(2+) paradox-induced changes in heart.

The effects of ischemic preconditioning (IP) on changes in cardiac performance and sarcoplasmic reticulum (SR) function due to Ca(2+) paradox were investigated. Isolated perfused hearts were subjected to IP (three cycles of 3-min ischemia and 3-min reperfusion) followed by Ca(2+)-free perfusion and reperfusion (Ca(2+) paradox). Perfusion of hearts with Ca(2+)-free medium for 5 min followed by reperfusion with Ca(2+)-containing medium for 30 min resulted in a dramatic decrease in the left ventricular (LV) developed pressure and a marked increase in LV end-diastolic pressure. Alterations in cardiac contractile activity due to Ca(2+) paradox were associated with depressed SR Ca(2+)-uptake, Ca(2+)-pump ATPase, and Ca(2+)-release activities as well as decreased SR protein contents for Ca(2+)-pump and Ca(2+) channels. All these changes due to Ca(2+) paradox were significantly prevented in hearts subjected to IP. The protective effects of IP on Ca(2+) paradox changes in cardiac contractile activity as well as SR Ca(2+)-pump and Ca(2+)-release activities were lost when the hearts were treated with 8-(p-sulfophenyl)-theophylline, an adenosine receptor antagonist; KN-93, a specific Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) inhibitor; or chelerythrine chloride, a protein kinase C (PKC) inhibitor. These results indicate that IP rendered cardioprotection by preventing a depression in SR function in Ca(2+) paradox hearts. Furthermore, these beneficial effects of IP may partly be mediated by adenosine receptors, PKC, and CaMK II.

Effects of p51/p63 missense mutations on transcriptional activities of p53 downstream gene promoters.

The p51/p63 gene is a homologue of p53, the product of which acts as a transcriptional activator by binding to p53-responsive elements in the promoter regions of several p53 downstream genes. Recently, we identified four distinct mutations in the p51/p63 gene after screening >200 human tumors and cell lines. Because all of the detected p51/p63 mutations were missense mutations, the pathogenic effect of these mutations is difficult to determine without performing a functional analysis. In this study, we examined the transcriptional activity of tumor-derived p51/p63 missense mutations using a yeast-based assay and compared the data with that of artificial p51/p63 missense mutations at residues corresponding to the positions and substituted residues of p53 mutation "hotspots." Although most of the p51/p63 missense mutations at the p53 hotspot residues were unable to transactivate the promoters used in this study, the tumor-derived p51/p63 missense mutations retained their ability to transactivate the MDM2 and/or the BAX promoter but not the p21/WAF1 promoter. These results suggest that the p51/p63 mutation might be involved in an unknown tumor suppression pathway distinct from that of p53.

[Biological inter- and intra-individual variations of serum immunochemical constituents and their allowable limits of analytical error].

The biological inter- and intra-individual variations of serum immunochemical constituents were estimated by the analysis of variance. Twelve samples taken at weekly intervals were analyzed for 22 healthy individuals. As for immunoglobulin (Ig) G, IgA, IgM, IgE, C-reactive protein, anti-streptolysin O, alpha-fetoprotein, complement component 3 and 4, and ferritin, the intra-individual variations (with coefficient of variations (CVs) ranging from 4.8% to 51.7%) were smaller than the inter-individual variations (with CVs ranging from 14.6% to 107.6%). The values for carcinoembryonic antigen (with CVs of inter- and intra-individual variation being 31.6% and 39.6%, respectively) and beta 2-microglobulin (with CVs of inter- and intra-individual variation being 14.4% and 13.1%, respectively) were similar. The biological variations of serum immunochemical constituents, examined in this study, were larger than those of serum chemical constituents. Based on our data, we propose allowable limits of analytical error, which is less than one-half of the average intra-individual variation for evaluation of imprecision and is less than one-quarter of the inter- plus intra-individual variation for evaluation of inaccuracy.

Status of Ca2+/calmodulin protein kinase phosphorylation of cardiac SR proteins in ischemia-reperfusion.

Although the sarcoplasmic reticulum (SR) is known to regulate the intracellular concentration of Ca2+ and the SR function has been shown to become abnormal during ischemia-reperfusion in the heart, the mechanisms for this defect are not fully understood. Because phosphorylation of SR proteins plays a crucial role in the regulation of SR function, we investigated the status of endogenous Ca2+/calmodulin-dependent protein kinase (CaMK) and exogenous cAMP-dependent protein kinase (PKA) phosphorylation of the SR proteins in control, ischemic (I), and ischemia-reperfused (I/R) hearts treated or not treated with superoxide dismutase (SOD) plus catalase (CAT). SR and cytosolic fractions were isolated from control, I, and I/R hearts treated or not treated with SOD plus CAT, and the SR protein phosphorylation by CaMK and PKA, the CaMK- and PKA-stimulated Ca2+ uptake, and the CaMK, PKA, and phosphatase activities were studied. The SR CaMK and CaMK-stimulated Ca2+ uptake activities, as well as CaMK phosphorylation of Ca2+ pump ATPase (SERCA2a) and phospholamban (PLB), were significantly decreased in both I and I/R hearts. The PKA phosphorylation of PLB and PKA-stimulated Ca2+ uptake were reduced significantly in the I/R hearts only. Cytosolic CaMK and PKA activities were unaltered, whereas SR phosphatase activity in the I and I/R hearts was depressed. SOD plus CAT treatment prevented the observed alterations in SR CaMK and phosphatase activities, CaMK and PKA phosphorylations, and CaMK- and PKA-stimulated Ca2+ uptake. These results indicate that depressed CaMK phosphorylation and CaMK-stimulated Ca2+ uptake in I/R hearts may be due to a depression in the SR CaMK activity. Furthermore, prevention of the I/R-induced alterations in SR protein phosphorylation by SOD plus CAT treatment is consistent with the role of oxidative stress during ischemia-reperfusion injury in the heart.

Differential roles of Akt, Rac, and Ral in R-Ras-mediated cellular transformation, adhesion, and survival.

Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.

Influence of p53 mutation on pathological grade, but not prognosis of non-Hodgkin's lymphoma.

Mutations in the p53 gene were detected in 27 of the 107 (25%) cases of non-Hodgkin's lymphoma (NHL), examined by assaying the transcriptional activity of p53 in yeast. A relatively high mutation rate of p53 was observed in B-cell intermediate-grade NHL and in T-cell high-grade immunoblastic NHL, in contrast to the relatively low mutation rate observed in other pathological classifications. However, retrospective analyses of all 76 cases revealed that the survival profile and therapeutic responses were very similar in NHL patients bearing lymphomas with a mutant p53 or with the wild-type p53 even within the subclasses characterized by frequent p53 mutation. In patients with high-intermediate grade tumors, the median survival period was 24 months in mutated p53 cases and 14 months in wild-type cases. Complete remission (CR) was observed in 9 of the 17 patients (53%) with mutated forms of p53 and 18 of the 35 patients (51%) with wild-type p53 genes. Our analyses of NHL patients revealed that the presence of p53 mutations may influence pathological grades of NHL, but did not strongly correlate with poor prognosis or reduced chemo/radiosensitivity in NHL. Hence, mutations of p53 do not serve as a prognostic, or chemo/radiosensitivity marker in NHL.