Inhibition of the membrane repair protein annexin-A2 prevents tumor invasion and metastasis.

Cancer cells are exposed to major compressive and shearing forces during invasion and metastasis, leading to extensive plasma membrane damage. To survive this mechanical stress, they need to repair membrane injury efficiently. Targeting the membrane repair machinery is thus potentially a new way to prevent invasion and metastasis. We show here that annexin-A2 (ANXA2) is required for membrane repair in invasive breast and pancreatic cancer cells. Mechanistically, we show by fluorescence and electron microscopy that cells fail to reseal shear-stress damaged membrane when ANXA2 is silenced or the protein is inhibited with neutralizing antibody. Silencing of ANXA2 has no effect on proliferation in vitro, and may even accelerate migration in wound healing assays, but reduces tumor cell dissemination in both mice and zebrafish. We expect that inhibiting membrane repair will be particularly effective in aggressive, poor prognosis tumors because they rely on the membrane repair machinery to survive membrane damage during tumor invasion and metastasis. This could be achieved either with anti-ANXA2 antibodies, which have been shown to inhibit metastasis of breast and pancreatic cancer cells, or with small molecule drugs.

E-cadherin and Snail Immunoexpression in Colorectal Adenocarcinomas.

Epithelial-mesenchymal transition (EMT) is a fundamental process which governs invasiveness. E-cadherin plays a major role in development, organogenesis and tissue formation, but also in tumor progression. Snail is a transcription factor described as a direct repressor of E-cadherin during development and in carcinogenesis. In this study we analyzed E-cadherin and Snail immunoexpression in 47 cases of colorectal adenocarcinoma (CRC) in comparison with some histopathological prognostic factors. The majority of cases were G2 tumors in stages II and III, with vascular and perineural invasion. All cases showed positive cytoplasmic signal for E-cadherin and Snail. E-cadherin reactions were intense with the highest composite score (CS) values in CRC G1. CS values of E-cadherin decreased with the advancement in tumor stage and the association with vascular and perineural invasion was statistically significant. Snail immunoreaction was intense with the highest values of CS in CRC G3, being more evident with the increase of tumor staging, aspect which was statistically significant. CS and Snail association demonstrated a statistically significant aspect related to vascular invasion. We found a negative linear correlation of E-cadherin and Snail expression. The obtained results indicate the implication of Snail and E-cadherin in EMT of CRC, aspect which is useful in the evaluation and stratification of patients with CRC for the targeted specific therapy.

Importance of Follow-Up and Early Detailed Evaluation in Early Onset Growth Restricted Fetuses.

Early onset fetal growth restriction (EO-FGR) is associated with significant feto-maternal complications, therefore efforts should be made to identify the causes and the potential outcome of the pregnancy. Some of the pitfalls in first-trimester imaging of the fetal anomalies are related to the inadequacy of the examination, because of the fetal position and limited clarity in relation to the size of the structures being examined. In this paper we present a case where careful ultrasound scan follow-up and the use of both approaches transabdominal and transvaginal were useful to complete a detailed structural evaluation as part of the diagnosis, management and prognosis of a fetuses diagnosed with EO-FGR in the first trimester and a triploidy with atypical ultrasound features.

Histopathological Prognostic Factors for Colic Adenocarcinoma.

The majority of colorectal carcinomas are adenocarcinomas derived from the colic mucosae cell, more frequently moderately differentiated. The purpose of this study was to determine de incidence of CRC and the relationship between histopathological risk factors in patients with colic adenocarcinomas. The study included 144 cases of CRC diagnosed within the Pathology Laboratory of the Clinical County Hospital of Craiova in the year 2017.The biological material consisted in samples from colectomies and hemicolectomies provided from patients admitted within the surgical clinics of the same hospital, then fixed with 10% buffered formalin and afterwards processed using the classic histopathological technique of paraffin inclusion and staining with hematoxylin and eosin. We observed certain histopathological parameters such as: pattern, grading, stage, vascular invasion and neural invasion. The mean age of diagnostic was 68.6 ± 11.2, and it was predominantly male patients (64.6%). Most cases presented with mucinous pattern (31.9%) and cribriform comedocarcinoma type (29.9%). The majority were classified as stage III B (34%), being moderately differentiated (64.6%) and associated with vascular invasion (47.2%) and perineural invasion (25.7%). Statistical analysis indicated significant relationships between tumor stage and differentiation grade (p<0.01, χ²test), as well as between tumor stage and vascular invasion (p<0.05, χ²test), without including perineural invasion (p<0.05, χ²test).

Histopathological Prognostic Factors in Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (CCRCC) are the most frequent type of renal cell carcinoma. Fuhrman grade and tumor stage are prognostic factors with great importance in survival rate. This study was performed on 75 cases of CCRCC diagnosed by the Anatomical Pathology Laboratory of the County Clinical Emergency Hospital of Craiova between 2014 and 2017. The biological material was represented by pieces of nephrectomy. The cases were analyzed on two criteria: epidemiology (age, sex) and histopathology (Fuhrman grade, tumor stage, architectural pattern, sarcomatoid transformation, and necrosis). Statistical analysis was done using Chi Square tests in IBM SPSS software. Average diagnosis age of CCRCC was 58.8±10.2 years, predominantly in male patients (66.7%). Tumor sizes were between 2 and 14cm, with an average of 6.7±2.9cm. Most cases were determined to be tumor stage III (60%) and Fuhrman grade 2 (56%), followed, in order of frequency, by tumor stages I and II (28% and 10.7%) and Fuhrman grades 3 and 1 (21.3% and 20%). High Fuhrman grade CCRCC were significantly associated with advanced tumor stage (p<0.05, χ2 test). Most cases presented a mixed pattern, significantly associated with advanced tumor stages (p<0.05, χ2 test). Even though the presence of sarcomatoid transformation was more frequent in advanced tumor stages, it wasn't significantly linked to them (p<0.05, χ2 test). Conclusions: Analyzed histopathological parameters are useful for determining CCRCC aggressiveness. CCRCC in advanced tumor stages is associated with high Fuhrman grade and mixed architectural pattern.

C-terminal cleavage of human Foxp3 at a proprotein convertase motif abrogates its suppressive function.

Foxp3 plays a critical role in the development and function of regulatory T cells (Tregs). Differences in translational and post-translational processing of murine and human Foxp3 have been recently reported. Human Foxp3 exists as four isoforms generated by alternative splicing. Mouse Foxp3 only exists as one isoform, but can be proteolytically cleaved by N-terminal and/or C-terminal proprotein convertase subtilisin/kexins (PCSKs). Here, we show by transcriptome analysis that the proprotein convertases PCSK7, PCSK5 and Furin are present in human CD4(+) T cells with different expression patterns. Notably, after in vitro activation, only PCSK7 and Furin are expressed in Tregs and T effector cells (Teffs), with overexpression of PCSK7 in Tregs compared to Teffs. Human Foxp3 protein displays specific motifs that can be potentially cleaved by convertases. Consequently, we transduced human CD4(+) cells with Foxp3-expressing lentiviral vectors and assessed the generation of proteolytically cleaved Foxp3 forms by Western blot. Three different Foxp3 forms were detected, indicating that human Foxp3 can also be subjected to proteolytic cleavage at the N-terminal and C-terminal ends. These results prompted us to assess the suppressive activity associated with each forms. We observed that full length and N-cleaved Foxp3-transduced CD4(+) T cells similarly suppressed the in vitro proliferation of Teffs. However, the C-cleaved or N&C-cleaved Foxp3 forms afforded almost no suppressive function, indicating a crucial role of the human Foxp3 C-terminal region in Tregs suppressive activity, in marked contrast with the report of a superior suppressive activity for the C-cleaved murine Foxp3 compared to the full length.

Pressure garment therapy (PGT) of burn scars: evidence-based efficacy.

Hypertrophic burn scars pose a challenge for burn survivors and providers. In many cases, they can severely limit a burn survivor's level of function, including work and recreational activities. A widespread modality of prevention and treatment of hypertrophic scarring is the utilization of pressure garment therapy (PGT). Despite the magnitude of the problem of hypertrophic scarring and the ubiquitous use of pressure garments as therapy, strong clinical evidence of the efficacy of PGT in the literature is lacking. Some of the challenges facing measurement of efficacy of PGT on hypertrophic scarring are lack of clear definitions for degree of hypertrophic scarring, inability to quantify pressure applied to scars, patient noncompliance to strict PGT time schedules, and inability to conduct randomized controlled trials comparing PGT to no therapy for ethical reasons since PGT is considered a standard of care. In this review, we attempt to summarize and analyze evidence-based literature on PGT and its efficacy in burn hypertrophic scars published in English language in the past 15 years.

Les cicatrices de brûlures hypertrophiques représentent un défi pour les survivants de brûlures et les fournisseurs. Dans de nombreux cas, ils peuvent gravement limiter le niveau de fonction d'un survivant de brûlure, y compris au travail et pendant les loisirs. Une modalité généralisée de la prévention et le traitement des cicatrices hypertrophiques est l'utilisation de la thérapie de vêtement de compression (TVC). Malgré l'ampleur du problème des cicatrices hypertrophiques et l'utilisation omniprésente de vêtements compressifs en tant que thérapie, dans la littérature il n'y a pas de preuves cliniques solides de l'efficacité de la TVC. Quelques-uns des défis auxquels fait face la mesure de l'efficacité de ce traitement sur les cicatrices hypertrophiques sont: le manque de définitions claires pour degré de cicatrisation hypertrophique, l'incapacité de quantifier la pression appliquée sur les cicatrices, la non-conformité des patients en ce qui concerne les horaires strictes du traitement, et l'incapacité de mener des essais comparatifs randomisés comparant cette thérapie à aucun traitement pour des raisons éthiques car la TVC est considérée comme une norme de soins. Dans cette revue, nous tentons de résumer et d'analyser la littérature fondée sur des preuves de la TVC et son efficacité dans les cicatrices hypertrophiques des brûlures publiés en langue anglaise au cours des 15 dernières années.

Inhibition of tumor cells proliferation and migration by the flavonoid furin inhibitor isolated from Oroxylum indicum.

BACKGROUND: The medicinally active plant Oroxylum indicum (OI) has drawn considerable research interest because of its many observed biological activities. Of particular interest is its antitumorigenic activity. The plant is a rich source of flavonoids and their glycosides. Recently flavonoids have been described as inhibitors of kexin-type proteases of superfamily Proprotein Convertase Subtilisin/ Kexins (PCSKs) which have been implicated in tumor growth and malignancy. These enzymes particularly furin (PCSK3) cleaves inactive precursor growth factors into their mature forms that promote tumor growth. As a result, finding furin-inhibitors became of high interest in cancer research. In this regard, the plant OI with known anticancer activities may provide an important source. OBJECTIVE: The objective of this study is to examine and compare anti-tumorigenic activity of furin inhibitory flavonoid compounds from OI. RESULTS: Studies were conducted to evaluate the effect on CT-26 cell proliferation and migration of 4 flavonoids baicalein, chrysin, oroxylin-A and its glycoside isolated from OI. Data revealed that baicalein exhibited most potent inhibitory effect on proliferation and migration on the analyzed tumor cell line. Baicalein at 10 µM completely blocked the proliferation even after 5 days. The results are consistent with the observed in vitro anti-furin activity of baicalein as measured against a fluorogenic peptide and pro-hVEGF-C as substrates. Mature VEGF-C is a strong indicator and biomarker of tumor progression and therefore the antifurin activity may explain the observed anticancer properties of baicalein. Since baicalein is the major constituent of OI, our data provided scientific rationale for the observed anticancer activity of OI and also offered a new lead molecule for future exploration as potential antitumor agents.

Thermal injury to the hand: review of the literature.

This paper is a comprehensive review of hand burn injuries. The different classifications of thermal burns, out- and inpatient care, indications for escharotomies as well as surgical management, skin substitutes, and paediatric hand burns are thoroughly reviewed.

Blockade of furin activity and furin-induced tumor cells malignant phenotypes by the chemically synthesized human furin prodomain.

Processing of cancer-associated precursor proteins such as growth factors by the Proprotein Convertase furin is an important cellular process for acquisition of malignant phenotype and metastatic potential of tumor cells. Furin is inhibited by its own prodomain protein which also plays role in the folding of mature furin. Herein, the complete 83-mer furin prodomain protein was chemically synthesized for the first time by solid phase peptide chemistry and its effects on furin activity and tumor cells malignant phenotypes were evaluated. It inhibited furin activity in a competitive manner with low nanomolar inhibition constant (Ki). Expression of furin prodomain cDNA in tumor cells or cells incubated with the corresponding protein blocked furin-cleavage of proPDGF-A. This was associated with significant reduction in tumor cells proliferation, migration as well as invasion. Interestingly shorter synthetic furin prodomain peptides derived from either its primary or secondary activation site alone weakly inhibited furin and displayed limited effects on tumor cells. This suggests that the combined presence of the above two prodomain segments is crucial for prodomain's potent furin-inhibitory and hence anticancer activities.

Regulation of prohepcidin processing and activity by the subtilisin-like proprotein convertases Furin, PC5, PACE4 and PC7.

BACKGROUND AND AIMS: Hepcidin is an iron homoeostasis regulator peptide. Loss-of-function mutations cause juvenile haemochromatosis while its over-expression results in anaemia. However, the mechanism and function of preprohepcidin conversion to mature hepcidins (25, 22 and 20 amino acid C-terminal peptides) are not well known. After removal of the signal peptide, the first proteolytic cleavage occurs within the basic motif RRRRR(59)DT, suggesting the involvement of proprotein convertase (PC) family members in this process. METHODS AND RESULTS: Using cell transfection experiments, the processing of preprohepcidin in the human hepatocyte line Huh-7 was found to be inhibited by the Furin inhibitors serpin alpha1-antitrypsin (alpha1-PDX) and prosegment preproFurin (ppFurin). Site-directed mutagenesis analysis confirmed the RRRRR(59)DT preprohepcidin cleavage site. In parallel, the lack of preprohepcidin processing found in the PC activity-deficient cell line LoVo was restored by the expression of Furin, paired basic amino acid cleaving enzyme 4 (PACE4), PC5 or PC7. This finding is consistent with the in vitro digestions of a synthetic peptide mimicking the cleavage site of preprohepcidin. In addition, during mouse embryonic development the major expression of hepcidin found in the liver coincided with that of Furin. While hepcidin induces the degradation of the iron transporter ferroportin, its RRRRR(59) to SSSSS(59) mutant is not active. CONCLUSIONS: These results demonstrate the key role of the convertases Furin, PACE4, PC5 and/or PC7 in the generation and secretion of active hepcidin and suggest that the control of hepcidin processing as a potential therapeutic/diagnostic strategy in hepcidin-related disorders such as haemochromatosis, inflammatory diseases, anaemia and cancer.

Articular chondrocyte aging and endothelin-1.

OBJECTIVE: To compare in cell culture endothelin-1 (ET-1) production, receptor density, and effect on macromolecular synthesis by articular chondrocytes (AC). METHODS: AC were isolated from 1-month and 18-month old rats and cultured as monolayers. They were incubated with ET-1 without or with iNOS inhibitors, nitro-L-arginine methyl ester (L-NAME) or guanylate cyclase inhibitor, LY83583 and then [3H]thymidine, 35SO4 and [3H]proline incorporations were measured. The density and affinity for 125I-ET-1 of binding sites, and receptor isotypes were determined. The cells were also treated with interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha), and then ET-1 productions measured. As well, the cells were challenged with NOC-5 (nitric oxide donor) or ET-1 and then ET-1 and NO respectively were measured. RESULTS: A concentration-dependent stimulation of DNA, PG, collagen and NO synthesis was obtained when cells were incubated with ET-1 for 24-h. Eighteen-month old chondrocytes incorporated per microg DNA more [3H]thymidine, 35SO4 and [3H]proline but less NO when challenged with ET-1 than the 1-month old cells. However, strong inhibition of this initial stimulation was seen after 48-h. L-NAME and LY83583 enhanced basal-, and ET-1-induced initial stimulation and completely suppressed late (at 48-h to 72-h) ET-1-induced inhibition, suggesting NO was responsible for this inhibitory effect. Eighteen-month old chondrocytes expressed per mug DNA more high affinity receptors of predominantly ET(A) subtype. They also produced more ET-1 but less NO under basal conditions and more ET-1 when challenged with IL-1beta and TNF-alpha. NOC-5 inhibited the production of ET-1. CONCLUSIONS: Eighteen-month old chondrocytes produce more ET-1, possess more ET-1-specific receptors, and increase more DNA, PG and collagen synthesis when challenged during 24-h with ET-1. NO, which suppresses ET-1 production and the production of which is increased by ET-1, seems to account for the late ET-1-induced inhibition of macromolecular synthesis. The possible implication of ET-1 in aging as related to osteoarthritis is discussed.

Human synovium produces substances that inhibit DNA and stimulate proteoglycan and collagen synthesis by cultured human articular chondrocytes and synovial fibroblasts.

The effects of synovial conditioned medium (SCM) on DNA, proteoglycan (PG), and protein-collagen synthesis and respective gene expressions, in human articular chondrocytes (AC) and DNA synthesis in synovial fibroblasts (SFb), were studied in monolayer culture. All SCM exhibited concentration-dependent inhibition of [3H]thymidine incorporation in both AC and SFb. In contrast, SCM from three OA patients stimulated [35S]SO4 and [3H]glycine incorporations and the expression (RT-PCR) of aggrecan- and type II collagen-specific mRNAs in AC. The production of agents that inhibit DNA synthesis was blocked by indomethacin and dexamethasone and stimulated by IL-1 beta and TNF-alpha. The inhibitory substances were not produced by heat-inactivated tissue nor cultured SFb or AC and were completely solubles in methanol. It is postulated that synovial tissue secretes lipids, most probably arachidonic acid metabolites. These may counteract growth of an inflammatory synovial pannus by inhibiting SFb proliferation and enhance repair of damaged tissues by stimulating the matrix synthesis.

Mechanism of inhibition of endothelin-1-stimulated proteoglycan and collagen synthesis in rat articular chondrocytes.

The aim of this study was to determine the effects of endothelin-1 (ET-1) on proteoglycan (PG) and collagen synthesis by rat articular chondrocytes (RAC). PG and collagen synthesis was measured by [(35)S]-sulphate and [(3)H]-glycine incorporation, respectively into monolayers of confluent RAC exposed to ET-1 (10(-11) M-10(-7) M). ET-1 stimulated PG and collagen synthesis in these cells in a concentration-dependent manner during the first 24 h of incubation. Prolonged contact of the cells with ET-1 resulted in a gradual decrease, and finally, inhibition of ET-1 effects. This inhibition is mediated by nitric oxide (NO) released in response to ET-1 since: (1) nitric oxide synthase inhibitor, nitro-L-arginine-methyl ester (L-NAME), enhanced both basal and ET-1-induced [(35)S]-sulphate and [(3)H]-glycine incorporations; (2) sodium nitroprusside (SNP), which spontaneously releases NO, inhibited both basal and ET-1-induced incorporations, and was also able to suppress the effects of L-NAME; (3) NO levels in the culture media were also correlated with the inhibition of [(35)S]-sulphate and [(3)H]-glycine incorporation; and (4) SNP also inhibited aggrecan and collagen II transcriptions, probably via cGMP. This effect was mimicked with 8-bromo-cGMP. Interestingly, the LY83583, which blocks the NO-dependent production and release of cGMP, inhibited PG-collagen synthesis but had no effect on their mRNA expressions. Thus, normal levels of cGMP appeared to be necessary for PG-collagen synthesis, whereas decreased levels are detrimental. In conclusion, NO, produced by rat AC in response to ET-1, counteracts the stimulation and finally induces inhibition of PG-collagen synthesis by ET-1 in these cells but NO-induced cGMP is only partially responsible for this inhibition.

Granulocyte-macrophage colony stimulating factor activates proteoglycan, type II collagen, and cAMP production by rat articular chondrocytes through specific binding sites.

OBJECTIVE: To evaluate the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on rat articular chondrocyte (AC) with respect to DNA synthesis, collagen type II and proteoglycan (PG) synthesis and expression, and cAMP production; to examine these cells for the presence of GM-CSF-specific binding sites; and to study their regulation by growth factors and cytokines. METHODS: First passage monolayers of rat AC were incubated with various concentrations of recombinant human GM-CSF, and then [3H]-thymidine, [3H]-proline, and [35S]SO4 incorporation and cAMP production were measured. The density of GM-CSF-specific binding sites, the effects of growth factors and cytokines on receptor density, and the activation of certain post-receptor signaling pathways were also examined by labeling the cell monolayers with [125I]-GM-CSF. RESULTS: GM-CSF (6-100 U/ml) inhibited (30%) [3H]-thymidine incorporation into DNA, and, in contrast, stimulated up to 3.6- and 2-fold [35S]SO4 and [3H]-proline incorporation into glycosaminoglycan side chains and collagen molecules, respectively. GM-CSF also increased aggrecan and type II collagen (Coll II) transcripts by 2- to 3-fold, respectively. These effects were associated with a concentration-dependent increase in cAMP production. A single class of high affinity (Kd = 98 pM; Bmax = 7.08 pM/microg DNA) binding sites of about 220 kDa were found. The [125I]-GM-CSF binding to the cells was slightly increased with phorbol 12-myristate 13-acetate (PMA), insulin-like growth factor-I, platelet derived growth factor, basic fibroblast growth factor, and tumor necrosis factor-alpha, and decreased with pertussis toxin, cholera toxin, and interleukin-1beta. CONCLUSION: These results suggest that GM-CSF may play a role in the regulation of chondrocyte metabolism as an anabolic agent and may stimulate cartilage healing under pathological conditions.

Cooperative regulation of the invasive and metastatic phenotypes by different domains of the type I insulin-like growth factor receptor beta subunit.

The receptor for the type 1 insulin-like growth factor (IGF-I) regulates multiple cellular functions impacting on the metastatic phenotype of tumor cells, including cellular proliferation, anchorage-independent growth, survival, migration, synthesis of the 72-kDa type IV collagenase and invasion. We have used site-directed mutagenesis to generate domain-specific mutants of the receptor beta subunit to analyze the role of specific tyrosines in the regulation of the invasive/metastatic phenotype. Poorly invasive M-27 carcinoma cells expressing low receptor numbers were transfected with a plasmid vector expressing IGF-I receptor cDNA in which single or multiple tyrosine codons in the kinase domain, namely Tyr-1131, Tyr-1135, and Tyr-1136 or the C-terminal tyrosines 1250 and 1251 were substituted with phenylalanine. Changes in the invasive and metastatic properties were analyzed relative to M-27 cells expressing the wild type receptor. We found that cells expressing the Y1131F,Y1135F,Y1136F or Y1135F receptor mutants lost all IGF-IR-dependent functions and their phenotypes were indistinguishable from, or suppressed relative to, the parent line. The Y1250F,Y1251F substitution abolished anchorage-independent growth, cell spreading, and the anti-apoptotic effect of IGF-I whereas all other IGF-IR-dependent phenotypes were either unperturbed (i.e. mitogenicity) or only partially reduced (migration and invasion). The results identify three types of receptor-dependent functions in this model: those dependent only on an intact kinase domain (DNA synthesis), those dependent equally on kinase domain and Tyr-1250/1251 signaling (e.g. apoptosis, soft agar cloning) and those dependent on kinase domain and enhanced through Tyr-1250/1251 signaling (migration, invasion). They suggest that signals derived from both regions of the receptor cooperate to enhance tumor metastasis.

Inhibition of proprotein convertases is associated with loss of growth and tumorigenicity of HT-29 human colon carcinoma cells: importance of insulin-like growth factor-1 (IGF-1) receptor processing in IGF-1-mediated functions.

Proprotein convertases (PCs) of the subtilisin/kexin family are responsible for the activation of prohormones, protrophic factors, and their receptors. We sought to determine whether loss of PC-mediated activities might affect the malignant phenotypes of cancer cells. Stable transfectants of alpha(1)-antitrypsin Portland (alpha(1)-PDX) cDNA, coding for a potent PC inhibitor, were analyzed in model HT-29 cells (HT-29/PDX) and in other cell lines. Expression of alpha(1)-PDX resulted in a proinsulin-like growth factor-1 receptor (pro-IGF-1R) processing blockade, hence inhibiting the ability of exogenous IGF-1 to induce tyrosine phosphorylation of its beta-subunit and insulin-related substrate-1. Coexpression of IGF-1R with four different PCs or the novel convertase SKI-1 in the furin-defective LoVo-C5 cells demonstrated that pro-IGF-1R ( approximately 200 kDa) cleavage into IGF-1R (beta-subunit, approximately 105 kDa) can be achieved by furin and PC5A, but not by PACE4, PC7, or SKI-1. Expression of alpha(1)-PDX resulted in reduction of DNA synthesis and in anchorage-independent growth. Following serum deprivation, the alpha(1)-PDX transfectants exhibited an enhanced apoptotic phenotype and were insensitive to IGF-1-mediated [(3)H]thymidine incorporation and protection against apoptosis. These cells showed reduced invasiveness that paralleled decreased mRNA levels of urokinase-type plasminogen activator and its receptor, tissue-type plasminogen activator, and plasminogen activator inhibitor-1. Comparative subcutaneous inoculation of cells in nude mice revealed that animals injected with HT-29/PDX cells exhibited delayed and lower incidence of tumor development as well as reduced tumor size. Immunohistochemical analysis of CD31 antigen expression, a marker of endothelial cells, revealed reduced HT-29/PDX tumor vascularization. These findings indicate that PCs actively contribute to the growth and malignant phenotypes of HT-29 tumors, suggesting that PC inhibition strategies may be a useful adduct to the arsenal of colorectal anticancer gene therapies.

Regulation of urokinase plasminogen activator/plasmin-mediated invasion of melanoma cells by the integrin vitronectin receptor alphaVbeta3.

The integrin vitronectin receptor alphavbeta3 is a mediator of cellular migration and invasion and has been identified as a marker of progression in malignant melanoma. Using a human melanoma model, we have previously shown that this receptor was coordinately expressed with the receptor for the urokinase plasminogen activator (uPAR). In our present study, the link between these receptors was further investigated by assessing the effect of alphavbeta3 ligation on uPAR transcription and function. Using the reverse transcription-polymerase chain reaction, we found that receptor ligation by immobilized monoclonal antibodies (MAbs) induced a rapid increase (up to 4.5 fold) in uPAR mRNA levels, which was maximal 4 hr after cell attachment. An increase was also noted in plasminogen activator inhibitor type-1 (PAI-1) mRNA levels (2.7-fold), but none was noted in uPA levels. In addition, ligation of alphavbeta3 resulted in a significant increase in cell surface-associated plasmin levels, which coincided with a 2- to 3-fold increase in cell invasion as measured in the Matrigel invasion assay. This increase in invasion could in turn be abolished by antibodies directed to uPA and uPAR and by the plasmin inhibitors epsilon-aminocaproic acid and aprotinin. Furthermore, ligation of the integrin alphavbeta3 triggered a rapid increase of up to 12-fold in total cellular PKC activity, and this coincided with the redistribution of PKCbeta, but not PKCalpha, from the cytosol to the membrane. Treatment of the cells with the PKCbeta-specific inhibitor LY379196 blocked uPAR and PAI-1 mRNA induction and reduced the increase in cell invasion due to alphavbeta3 ligation, confirming the involvement of this isoform in the response. The results provide evidence that the vitronectin receptor can enhance invasion by regulating the uPAR/uPA/plasmin system of proteolysis and implicate PKCbeta as an intermediate in the activation pathway.

Articular chondrocytes from aging rats respond poorly to insulin-like growth factor-1: an altered signaling pathway.

This study investigates the effect of insulin-like growth factor-1 (IGF-1) and phorbol 12-myrystate 13-acetate (PMA) on 3H-thymidine, 35SO(4) and 3H -glycine incorporations, adenosine 3':5'-cyclic monophosphate (cAMP) production and protein kinase C (PKC) activation in cultured rat articular chondrocyte monolayers (RACM) derived from animals of different ages. It was found that IGF-1 stimulates all these cellular functions in cultures derived from all age groups in a concentration dependent manner, although the cells from 14-month old animals responded poorly. IGF-1 also induces in cells from 1-month old rats an increase in the expression of mRNAs specific for aggrecan and type II collagen molecules as shown with RT-PCR. These effects are mediated via IGF-1 interaction with specific receptors because the monoclonal antibody against the receptor protein suppresses more than 60% of the ligand-induced DNA synthesis. PMA, a direct PKC activator, potentiated IGF-1-induced effects in all cells but much more strongly in cells from young than in cells from 14-month old animals. The age-related failure of RACM to respond adequately to IGF-1 was correlated with a decrease in IGF-1-induced cAMP production, and IGF-1-induced and PMA-induced PKC activations. These results show that IGF-1 regulates the synthesis of DNA, proteoglycans (PG) and collagen II at the level of transcription and suggest that the reduced response of cell monolayers derived from 14-month old rats to IGF-1 is probably due to a failure of old cells to adequately transduce IGF-1 receptor-generated downstream signaling.

Endothelin-1 in monolayer cultures of articular chondrocytes from young and old rats: regulation by growth factors and cytokines.

The endothelin-1 (ET-1) concentrations were measured by RIA in the media of confluent monolayer cultures of rat articular chondrocyte (RAC) exposed to fetal calf serum (FCS) and several growth factors and cytokines. The cells were obtained from 1- and 18-month-old rats. First passage cells were starved in Dulbecco's modified Eagle's medium (DMEM) containing 0.2% FCS serum for 24 h and then incubated for 48 h in the same fresh medium with each of the following factors: fetal calf serum (FCS), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), and NO donor, sodium nitroprusside (SNP). The following was found: the cells from 18-month-old animals accumulated about twice as much ET-1 per microg DNA under basal (low serum) and stimulated conditions as cells from young rats. All, but PDGF and SNP produced concentration-dependent rise in ET-1 levels, the most effective being 10% FCS, IL-1beta, TNF-alpha, EGF, IGF-1 and LPS. TGF-beta caused the smallest stimulation and PDGF was ineffective or slightly inhibitory at high concentrations. SNP caused concentration-dependent decrease of ET-1 concentrations. ET-1-specific mRNA was identified by RT-PCR in cells incubated with the above factors and its concentration paralleled that of the peptide. This suggests that ET-1 found in the culture media of RAC stems, at least in part, from the synthesis. Increased immunoreactive peptide concentration and mRNA expression with the age of the donor rat and its regulation by several growth factors and cytokines suggest the involvement of ET-1 in chondrocytes' physiology and possibly pathology.