ERp29 regulates response to doxorubicin by a PERK-mediated mechanism.

ERp29 is an endoplasmic reticulum (ER) luminal protein with a putative secretion factor/escort chaperone function. Accumulated evidence has implicated ERp29 in the thyroglobulin secretion, polyoma virus transport and recently in carcinogenesis. ERp29 levels were elevated in the tumors of various origins and under the conditions of genotoxic stress, such as ionizing radiation. Here we report the induction of ERp29 during the treatment of cells with doxorubicin, a commonly used antineoplastic agent. Experiments in the p53 -/- cells and p53 knockout mouse revealed that doxorubicin effect on ERp29 is p53 dependent. The increase of ERp29 level appears to activate a negative feedback loop where the elevated amounts of ERp29 augment cell viability as shown by a clonogenic cell survival assay. To elucidate the mechanisms behind the doxorubicin effects we have studied the impact of ERp29 on the interaction with the ER stress-activated eukaryotic translation initiation factor 2-alpha kinase 3 (PERK) that was shown to facilitate tumor cells' resistance to drug toxicity. Co-immunoprecipitation demonstrated physical interaction of ERp29 with PERK and moreover, overexpression of ERp29 enhanced endogenous levels of PERK. Our results identify ERp29 as a novel regulator of PERK and provide evidence for the role of ER resident factors in the regulation of chemotherapeutic efficacy. These findings show that PERK may represent a nodal point between ER stress and chemotherapeutic response.

Targeted degradation of c-Fos, but not v-Fos, by a phosphorylation-dependent signal on c-Jun.

The proto-oncogene products c-Fos and c-Jun heterodimerize through their leucine zippers to form the AP-1 transcription factor. The transcriptional activity of the heterodimer is regulated by signal-dependent phosphorylation and dephosphorylation events. The stability of c-Fos was found to also be controlled by intracellular signal transduction. In transient expression and in vitro degradation experiments, the stability of c-Fos was decreased when the protein was dimerized with phosphorylated c-Jun. c-Jun protein isolated from phorbol ester-induced cells did not target c-Fos for degradation, which suggests that c-Fos is transiently stabilized after stimulation of cell growth. v-Fos protein, the retroviral counterpart of c-Fos, was not susceptible to degradation targeted by c-Jun.

EGFR, HER-2 and COX-2 levels in colorectal cancer.

AIMS: Receptor tyrosine kinases epidermal growth factor receptor (EGFR) and HER-2 and cyclooxygenase-2 (COX-2) are promising molecular targets for cancer therapy and/or prevention. The aim was to evaluate EGFR, HER-2 and COX-2 mRNA and protein expression in colorectal cancer patients. METHODS AND RESULTS: EGFR, HER-2 and COX-2 protein levels were evaluated by immunohistochemistry in malignant tissue, dysplastic tissue and normal mucosa samples from 124 cases with primary colorectal carcinoma. Moreover, the corresponding mRNA levels were assessed by quantitative reverse transcriptase-polymerase chain reaction in 46 colorectal carcinomas. There was strong correlation between mRNA and protein expression for EGFR (P < 0.001), HER-2 (P < 0.004) and COX-2 (P < 0.007). EGFR levels did not correlate with stage of the disease or tumour differentiation. HER-2 and COX-2 levels increased in advanced stages and in differentiated carcinomas. Furthermore, a correlation between HER-2 and COX-2 expression was revealed in neoplastic tissue. CONCLUSIONS: EGFR as well as HER-2 and COX-2 overexpression represent important alterations that are related to the molecular pathways underpinning colorectal carcinogenesis. Further investigation is required to evaluate the impact of these markers on the management of patients with colorectal cancer.

Mechanical loading triggers specific biochemical responses in mandibular condylar chondrocytes.

The effect of mechanical loading on the phosphorylation state of condylar cartilage proteins was investigated by high resolution electrophoretic analysis of 32P-labelled proteins from mechanically stimulated rat mandibular condylar chondrocytes. Specific dephosphorylation (and/or loss) of an acidic, 35-36 kDa protein(s) and of proteins overlapping with members of the ras superfamily of small GTP-binding proteins was observed. These responses may constitute part of a mechanically induced transduction system which establishes the differentiated phenotype.

Rab and rho GTPases are involved in specific response of periodontal ligament fibroblasts to mechanical stretching.

To investigate the contribution of ras-related signalling molecules to the mechanotransduction process, stretch-sensing human periodontal ligament (PDL) fibroblasts were isolated and cultured in dishes with a flexible bottom. The cells were stimulated by stretching the bottom of the dishes and membrane fractions were prepared and analysed at the level of mapping small GTP-binding proteins by high resolution two-dimensional gel electrophoresis followed by renaturing transfer and an [alpha-32P]GTP-overlay procedure. This analysis revealed that mechanically-stretched PDL fibroblasts exhibit complete down-regulation of rhoA and induction of rab6, rab17 and a putative member of the rab3 subfamily in a cell type-specific manner.

Novel biological agents for the treatment of hormone-refractory prostate cancer (HRPC).

Hormone-refractory prostate cancer (HRPC) is an inevitable evolution of prostate carcinogenesis, through which the normal dependence on hormones for growth and survival is bypassed. Although advances in terms of symptoms palliation and quality of life improvement have been addressed with current treatment options, innovative approaches are needed to improve survival rates. A thorough understanding of HRPC-associated molecular pathways and mechanisms of resistance are a prerequisite for novel potential therapeutic interventions. Preclinical and early clinical studies are ongoing to evaluate new therapies that target specific molecular entities. Agents under development include growth factor receptor inhibitors, small molecules targeting signal transduction pathways, apoptosis and cell-cycle regulators, angiogenesis and metastasis inhibitors, differentiation agents, telomerase inactivators, and epigenetic therapeutics. Incorporation of these agents into existing treatment regimens will guide us in the development of a multidisciplinary treatment strategy of HRPC. This article critically reviews published data on new biological agents that are being tested in HRPC clinical trials, highlights ongoing research and considers the future perspectives of this new class of agents.

Molecular 'palpation' of BPH: a tale of MAPK signalling?

Benign prostatic hyperplasia (BPH) is a very common cause of hospitalization and surgery is currently the most effective therapy. MAP kinases (MAPKs) are a group of protein kinases with an important function in integrating physiological and pathological stimuli that might impact on cellular growth, differentiation and programmed cell death (apoptosis). Certain components of the MAPK signal-transduction pathways are involved in stimulus-specific fine-tuning of the activities mediated by the various MAPK families. As homeostasis is impaired in the hyperplastic prostate, aberrant coordination of the MAPK cascades might be implicated in a proliferative-apoptotic imbalance. Here, we hypothesize that the pathogenesis of BPH might be facilitated by functional anomalies in the MAPK circuitry and postulate that pharmacological 'rewiring' of MAPK pathways offers a potentially exciting new avenue for improved therapeutic control of clinical BPH.

Type 2 diabetes mellitus and worm longevity: a transcriptional link to cure?

In Caenorhabditis elegans, an insulin-like signalling pathway culminates in a transcription factor (TF) that is homologous to a subfamily of TFs responsible for the regulation of a subset of insulin-responsive genes in humans. Under harsh conditions, C. elegans reduces signalling through this pathway and arrests developmentally in a manner that is similar to the metabolic syndrome of humans. We propose that an understanding of this pathway could lead to drugs with optimal potency and selectivity in the treatment of type 2 diabetes mellitus.

Serine phosphorylation of insulin receptor substrate-1: a novel target for the reversal of insulin resistance.

Insulin resistance, the failure to respond to normal circulating concentrations of insulin, is a common state associated with obesity, aging, and a sedentary lifestyle. Compelling evidence implicates TNFalpha as the cause and link between obesity and insulin resistance. Serine phosphorylation of insulin receptor substrate-1 seems prominent among the mechanisms of TNFalpha-induced insulin resistance. Recent advances indicate that serine kinases may phosphorylate and thus inhibit the tyrosine phosphorylation of insulin receptor substrate-1, revealing an integration point of TNFalpha and insulin signaling pathways. Selective targeting of the molecular scenery whereby this key phosphorylation occurs/operates represents a rich area for the development of rationally designed new antidiabetic drugs. In relation to efficacy and side effects, this prospect should permit a more precise and perhaps individualized approach to therapeutic intervention, allowing clinicians to focus the attack where the problem lies.

Mechanical stress induces DNA synthesis in PDL fibroblasts by a mechanism unrelated to autocrine growth factor action.

Periodontal regeneration is thought to require the proliferation of stress-sensitive periodontal ligament (PDL) fibroblast cells. The influence of physiological amounts of mechanical stretching on the DNA synthesis potential of human PDL fibroblasts was examined by means of an established, simple in vitro system of stretch application. A significant increase in the relative levels of incorporation of tritiated thymidine was observed in cultures stretched for 1-6 h. Neutralising antibodies for platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) did not blunt the DNA synthesis induction. This mitogenic response to stretch appears to be independent of an autocrine mechanism involving growth factors in general, because stretch-conditioned medium, when transferred to non-stretched fibroblasts, did not mimic the mitogenic effect of stretch.

Selective uptake and degradation of c-Fos and v-Fos by rat liver lysosomes.

The transcription factor c-Fos is a short-lived protein and calpains and ubiquitin-dependent systems have been proposed to be involved in its degradation. In this report, we consider a lysosomal degradation pathway for c-Fos. Using a cell-free assay, we have found that freshly isolated lysosomes can take up and degrade c-Fos with high efficiency. v-Fos, the oncogenic counterpart of c-Fos, can also be taken up by lysosomes, yet the amount of incorporated protein is much lower. c-Fos uptake is independent of its phosphorylation state but it appears to be regulated by dimerization with differentially phosphorylated forms of c-Jun, while v-Fos escapes this regulation. Moreover, we show that c-Fos is immunologically detected in lysosomes isolated from the liver of rats treated with the protease inhibitor leupeptin. Altogether, these results suggest that lysosomes can also participate in the selective degradation of c-Fos in rat liver.

Prostate cancer: horizons in the development of novel anti-cancer strategies.

Prostate cancer is the most common cause of non-cutaneous cancer in men and a leading lethal malignancy with increasing incidence worldwide. Inevitably, all patients will develop androgen-independent disease, which remains the main obstacle to improving survival. Unfortunately, existing first and second line hormonal treatment are condemned to extent survival for only a few years. These sobering data have forced researchers to start exploring in depth the molecular mechanisms implicated in the emergence of androgen independence and of prostate cancer invasion. In this vein, epoch-making discoveries in molecular oncology along with rapid expansion of our knowledge concerning the processes that govern differentiation, apoptosis, immune surveillance, angiogenesis, metastasis, cell cycle and signal transduction control, have unveiled a plethora of specific targets for prostate cancer gene therapy, immunotherapy and anti-invasion therapy, along with a variety of small-molecule compounds which inhibit or stimulate these pathways. These new anti-cancer approaches are in various stages of clinical development, providing exciting perspectives for the future of prostate cancer cure and enriching the current anti-cancer drug quiver with a new spectrum of therapeutic agents. The present review, through extensive literature cross-examination, discusses several novel anti-prostate cancer strategies, emphasizing on approaches that potentially may extend end-stage patient's survival.

Activation of the JNK-AP-1 signal transduction pathway is associated with pathogenesis and progression of human osteosarcomas.

Osteosarcomas represent the most common primary malignant bone tumors; however, comprehension of the molecular mechanisms underlying their pathogenesis is far from thorough. Studies in cultured cells have demonstrated that the c-Jun N-terminal kinase (JNK) signal transduction pathway participates in the proliferation, differentiation, and apoptosis of osteoblasts. Phosphorylated JNKs activate the oncoprotein c-Jun, which is known to form the activator protein-1 (AP-1) transcription factor as a homo- or heterodimer. c-Jun's principal dimerization partner is c-Fos, which participates in the differentiation and function of osteoblasts and in the pathogenesis of osteosarcomas. A similar role for the JNK cascade in the malignant transformation of human osteoblasts and in the generation of osteosarcomas has not been documented. Our study addressed the possibility that a functional upregulation of the JNK pathway is implicated in the pathogenesis of osteosarcomas. To this end, we employed immunohistochemistry to examine normal bone and osteosarcoma cells in paraffin-embedded sections from 56 patients with high-grade tumors and 15 patients with low-grade tumors. We assessed the protein levels of the two major JNK isoforms (JNK1 and JNK2); their phosphorylated-hence activated-species, p-JNK; their substrate, c- Jun; its phosphorylated (activated) form, pc-Jun; and c-Jun's heterodimeric partner, c-Fos. We also examined the immunohistochemical profile of the alpha chain of the nascent polypeptide-associated complex (alpha-NAC), an osteoblast-specific AP-1 coactivator that potentiates the transcriptional activity of the c-Jun/c-Jun homodimer. Positive immunostaining for JNK1, JNK2, p-JNK, c-Jun, pc-Jun, c-Fos, and alpha-NAC was observed in 86, 93, 94, 99, 97, 99, and 97.5% of the samples, respectively, whereas normal bone was devoid of these immunoreactivities. The cellular levels of all proteins were significantly correlated to each other (P < 0.001 for each correlation). Moreover, significantly higher expression levels of all proteins were detected in high-grade tumors compared to levels in low-grade ones. The observed expression profile of alpha-NAC implies that the active AP-1 in human osteosarcomas most likely comprises c-Jun/c-Jun homodimers. When cellular levels of the JNK pathway components and c-Fos were evaluated as possible biological markers of tumor grade, high expression of c-Jun and abundant pc-Jun predicted a high-grade tumor. Our findings provide novel evidence that the JNK signaling pathway is functionally operative in the malignant transformation of osteoblasts and the subsequent development and progression of human osteosarcomas. Evaluation of c-Jun expression and JNK-dependent activation may facilitate an improved prediction of the tumor's clinical behavior and potentially be exploited in designing patient-tailored treatment regimens.

Differential expression of retinoic acid receptor beta (RARbeta) and the AP-1 transcription factor in normal, premalignant and malignant human laryngeal tissues.

The anticancer effects of retinoids are mainly mediated by their nuclear receptors. Recent studies have demonstrated that retinoic acid receptor beta (RARbeta) plays a pivotal role from the early stages of laryngeal carcinogenesis; however, the exact mechanism of this detrimental effect has not yet been elucidated. One of the best-documented actions of retinoid receptors is the transrepression of activator protein-1 (AP-1) transcription factor activity, although this complex interplay has not been clarified. The present report is the first systematic morphological evaluation of the cross-talk of RARbeta and AP-1 transcription factor in a large series of human laryngeal tissues containing normal epithelium, premalignant lesions (hyperplasia and/or dysplasia) and squamous cell carcinoma. Immunohistochemical methodology was performed on formalin-fixed, paraffin-embedded sections by using a panel of monoclonal and polyclonal antibodies against RARbeta and the AP-1 components c-Jun, p-c-Jun (phosphorylated, active c-Jun) and c-Fos proteins. Their expression was screened and compared in 154 patients with various laryngeal histological entities. Nuclear expression of RARbeta, c-Jun, p-c-Jun and c-Fos was detected in 81 (89.2%), 48 (52.8%), 66 (72.6%) and 73 (80.3%), respectively, out of 91 specimens with normal-appearing laryngeal epithelium; in 86 (87.8%), 94 (95.9%), 94 (95.9%) and 94 (95.9%), respectively, out of 98 specimens with hyperplastic laryngeal epithelium; in 58 (56.8%), 92 (90.2%), 96 (94.1%) and 96 (94.1%), respectively, out of 102 specimens with dysplastic laryngeal epithelium; in 10 (22.3%), 41 (91.2%), 44 (97.8%) and 41 (91.2%), respectively, out of 45 specimens with well-differentiated squamous cell carcinoma; in 13 (30.3%), 37 (86%), 39 (90.7%) and 41 (95.3%), respectively, out of 43 specimens with moderately-differentiated squamous cell carcinoma; and in 8 (66.7%), 10 (83.3%), 12 (100%) and 12 (100%), respectively, out of 12 specimens with poorly-differentiated squamous cell laryngeal carcinoma. Statistical analysis and correlation of the intensity of nuclear immunostaining of the studied proteins among the various histological entities revealed statistically significant results. The progressive upregulation of the AP-1 transcription factor constituents and downregulation of the RARbeta protein detected from the onset of laryngeal tumorigenesis suggests an important role for the immediate-early AP-1/RARbeta on/off "switch" in the process of laryngeal carcinogenesis.

Oestrogen receptor beta (ERbeta) is abundantly expressed in normal colonic mucosa, but declines in colon adenocarcinoma paralleling the tumour's dedifferentiation.

Oestrogen Receptor beta (ERbeta) may protect against prostate and mammary cell proliferation and malignant transformation. Epidemiological studies indicate that oestrogens may reduce colon cancer risk. Since ERalpha is minimally expressed in normal and malignant colon, the aim of this study was to investigate the expression of ERbeta in both normal colonic wall and colon cancer. ERbeta expression was evaluated by immunohistochemistry in 90 cases of colon adenocarcinoma and nearby (>30-cm away) normal colonic wall, using a monoclonal antibody. Moderate or strong nuclear immunostaining was detected in superficial and crypt epithelium, endothelial cells, vascular smooth muscle cells, lymphocytes, enteric neurons and smooth muscular cells of the normal colonic wall. Superficial epithelial cells in normal colon demonstrated a significantly higher ERbeta expression than colon adenocarcinoma cells in both genders. The decline in ERbeta expression paralleled the loss of differentiation of malignant colon cells, regardless of the tumour's localisation. These findings suggest a protective role for ERbeta against colon carcinogenesis.

Preservation of protein phosphoryl groups in immunoprecipitation assays.

A phosphorylation-sensitive immunoprecipitation protocol is presented which ensures quantitative and qualitative recovery of phosphoantigens from different phosphatase environments. The protocol is based on the phosphatase-inactivating properties of a specific mixture of chemical compounds included in the "classical" immunoprecipitation buffers.

Induction of the CBP transcriptional co-activator early during laryngeal carcinogenesis.

PURPOSE: CREB-binding protein (CBP) is a transcriptional "integrator" that is suspected of contributing to tumorigenesis. This is the first systematic morphologic study evaluating CBP expression in a large series of human laryngeal tissues containing normal epithelium, premalignant lesions (hyperplasia and/or dysplasia), and squamous cell carcinoma. METHODS: Immunohistochemical methodology was performed on formalin-fixed paraffin-embedded sections by using a monoclonal anti-CBP antibody. CBP expression was screened and compared in 156 patients with various laryngeal histologic entities. RESULTS: Nuclear expression of CBP was found in 44 out of 91 (48.4%) specimens with normal-appearing epithelium (46.2% weak and only 2.2% moderate positivity), 92 out of 100 (92%) with hyperplastic lesions (56% weak, 36% moderate/strong, and only 8% no positivity), 80 out of 103 (77.7%) with dysplastic lesions (45.6% weak, 32.1% moderate/strong, and 22.3% no positivity), 37 out of 45 (82.2%) with well-differentiated carcinoma (42.2% weak, 40% moderate/strong, and 17.8% no positivity), 31 out of 43 (72.1%) with moderately differentiated carcinoma (32.6% weak, 39.5% moderate/strong, and 27.9% no positivity) and eight out of 12 (66.7%) with poorly differentiated carcinoma (41.7% weak, 25% moderate/strong, and 33.3% no positivity). Statistical analysis and correlation of the intensity of nuclear immunostaining among the various histologic entities revealed statistically significant results. CONCLUSIONS: Overexpression of CBP is detected from the very early stages of laryngeal carcinogenesis, suggesting that CBP may play a role in malignant transformation of precancerous laryngeal lesions. It is possible that overexpression of this protein is a prerequisite for the observed p53 upregulation in premalignant lesions, implying an indirect role of CBP in p53-mediated tumorigenic potential.

Androgen receptor (AR) expression is an independent unfavorable prognostic factor in gastric cancer.

PURPOSE: To investigate the expression of sex steroid receptors in gastric cancer and to correlate their tumor expression profile with the clinicopathological parameters and overall survival of the patients. METHODS: Immunohistochemical methodology was employed in formalin-fixed paraffin-embedded sections from 86 patients with gastric carcinoma. Monoclonal antibodies against androgen (AR), estrogen (ER), and progesterone (PR) receptors were used. Survival rates were estimated by the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis was performed by the Cox proportional hazards model. RESULTS: Fifteen (17.4%) cases of gastric adenocarcinomas were positive for AR, two (2.3%) were positive for PR and three (3.5%) were positive for ER. Significantly higher AR expression was found in tumors with metastases to lymph nodes (P = 0.03). Patients with AR-positive tumors (AR+) had worse prognosis than (AR-) patients (median survival 9 months vs 24 months, P = 0.03). Patients with AR- and heat shock protein 27 (HSP27)-positive tumors (AR+/HSP27+) had a median survival of 6 months, whereas (AR-/HSP27-) patients had a median survival of 42 months (P = 0.017). Multivariate analysis revealed that AR expression and UICC stage were independent factors of unfavorable prognosis (P = 0.037 and P = 0.0055, respectively). CONCLUSIONS: Identification of AR-positive gastric carcinomas in gastric biopsies may warrant a more aggressive therapeutic approach and anti-androgen or AR-targeted agents may represent a novel strategy in tackling this devastating malignancy.