Emanuela Marras

Protein kinase Cε is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway

We have shown previously that overexpression of the ε isoform of protein kinase C (PKCε) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/ε cells (overexpressing PKCε), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCε-transformed D/ε cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCε-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCε. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCε cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCε, and that overexpression of PKCε circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCε exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.

PKCδ acts as a growth and tumor suppressor in rat colonic epithelial cells

We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCδ, PKCε and PKCζ, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCζ and PKCε showed similar protein levels, PKCδ was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCδ was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCδ. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCδ was involved in growth control and transformation in this model, we overexpressed PKCδ in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCδ can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.

Use of the UPOINT Chronic Prostatitis/Chronic Pelvic Pain Syndrome Classification in European Patient Cohorts: Sexual Function Domain Improves Correlations

PURPOSE Patients with chronic prostatitis-chronic pelvic pain syndrome are difficult to treat due to the unknown etiology and complex clinical presentation. Clinical phenotyping may better correlate with multimodal treatment concepts than a current diagnosis. We evaluated a novel clinical phenotyping system in a database of patients with chronic prostatitis-chronic pelvic pain syndrome at 2 European institutions and correlated it with patient symptoms. We also investigated the addition of a sexual dysfunction domain in regard to symptom correlation and system internal consistency. MATERIALS AND METHODS We retrospectively classified 937 patients from Milan, Italy, and 290 from Giessen, Germany, with chronic prostatitis-chronic pelvic pain syndrome into a 6-domain phenotyping system, consisting of urinary, psychosocial, organ specific, infection, neurological and muscle tenderness domains, termed UPOINT. Symptom severity was assessed by the National Institutes of Health Chronic Prostatitis Symptom Index and the International Prostate Symptom Score. RESULTS There was significant correlation between the number of positive UPOINT domains and Chronic Prostatitis Symptom Index/International Prostate Symptom Score symptoms in the total and Italian cohorts but not in the German cohort. After adding a sexual dysfunction domain to create the modified UPOINTS system phenotypic domains also correlated significantly with Chronic Prostatitis Symptom Index symptoms in the German cohort. CONCLUSIONS Consistency of the UPOINT chronic prostatitis-chronic pelvic pain syndrome clinical phenotyping system was generally confirmed by our study and further refined by adding a sexual dysfunction domain. The treatment effect of clinical phenotyping with UPOINT(S) must be extensively evaluated in prospective treatment studies.

Polymorphisms in nonhomologous end‐joining genes associated with breast cancer risk and chromosomal radiosensitivity

As enhanced chromosomal radiosensitivity (CRS) results from non‐ or misrepaired double strand breaks (DSBs) and is a hallmark for breast cancer and single nucleotide polymorphisms (SNPs) in DSB repair genes, such as non homologous end‐joining (NHEJ) genes, could be involved in CRS and genetic predisposition to breast cancer. In this study, we investigated the association of five SNPs in three different NHEJ genes with breast cancer in a population‐based case‐control setting. The total patient population composed of a selected group of patients with a family history of the disease and an unselected group, consisting mainly of sporadic cases. SNP analysis showed that the c.2099‐2408G>A SNP (XRCC6) has a significant, positive odds ratio (OR) of 2.81 (95% confidence interval (CI): 1.30–6.05) for the heterozygous (He) and homozygous variant (HV) genotypes in the selected patient group. For the c.‐1310 C>G SNP (XRCC5) a significant OR of 1.85 (95%CI: 1.01–3.41) was found for the He genotype in the unselected patient group. On the contrary, the HV genotype of c.1781G>T (XRCC5) displays a significant, negative OR of 0.43 (95%CI: 0.18–0.99) in the total patient population. The He+HV genotypes of the c.2099‐2408G>A SNP (XRCC6) also showed high and significant ORs in the group of “radiosensitive,” familial breast cancer patients. In conclusion, our results provide preliminary evidence that the variant allele of c.‐1310C>G (XRCC5) and c.2099‐2408G>A (XRCC6) are risk alleles for breast cancer as well as CRS. The HV genotype of c.1781G>T (XRCC5) on the contrary, seems to protect against breast cancer and ionizing radiation induced micronuclei.

The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons

BackgroundA better understanding of the underlying mechanisms of DNA repair after low- and high-LET radiations represents a research priority aimed at improving the outcome of clinical radiotherapy. To date however, our knowledge regarding the importance of DNA DSB repair proteins and mechanisms in the response of human cells to high-LET radiation, is far from being complete.MethodsWe investigated the radiosensitizing effect after interfering with the DNA repair capacity in a human mammary epithelial cell line (MCF10A) by lentiviral-mediated RNA interference (RNAi) of the Ku70 protein, a key-element of the nonhomologous end-joining (NHEJ) pathway. Following irradiation of control and Ku-deficient cell lines with either 6 MV X-rays or p(66)+Be(40) neutrons, cellular radiosensitivity testing was performed using a crystal violet cell proliferation assay. Chromosomal radiosensitivity was evaluated using the micronucleus (MN) assay.ResultsRNAi of Ku70 caused downregulation of both the Ku70 and the Ku80 proteins. This downregulation sensitized cells to both X-rays and neutrons. Comparable dose modifying factors (DMFs) for X-rays and neutrons of 1.62 and 1.52 respectively were obtained with the cell proliferation assay, which points to the similar involvement of the Ku heterodimer in the cellular response to both types of radiation beams. After using the MN assay to evaluate chromosomal radiosensitivity, the obtained DMFs for X-ray doses of 2 and 4 Gy were 2.95 and 2.66 respectively. After neutron irradiation, the DMFs for doses of 1 and 2 Gy were 3.36 and 2.82 respectively. The fact that DMFs are in the same range for X-rays and neutrons confirms a similar importance of the NHEJ pathway and the Ku heterodimer for repairing DNA damage induced by both X-rays and p(66)+Be(40) neutrons.ConclusionsInterfering with the NHEJ pathway enhanced the radiosensitivity of human MCF10A cells to low-LET X-rays and high-LET neutrons, pointing to the importance of the Ku heterodimer for repairing damage induced by both types of radiation. Further research using other high-LET radiation sources is however needed to unravel the involvement of DNA double strand break repair pathways and proteins in the cellular response of human cells to high-LET radiation.

Effect of HIF-1 modulation on the response of two- and three-dimensional cultures of human colon cancer cells to 5-fluorouracil.

Tumour hypoxia represents a major obstacle to the success of radiotherapy and chemotherapy. The discovery that the hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular response to low oxygen led to the concept that inhibiting HIF-1 activity may sensitise hypoxic cancer cells to radiation and cytotoxic drugs. In the present study we investigate the effects of HIF-1 modulation on the response of the human colon adenocarcinoma cell line HCT116 to 5-fluorouracil (5FU). Increasing HIF-1 activity, either by exposing cells to hypoxia or by forced expression of a degradation-resistant form of HIF-1alpha, results in poor cell response to 5FU; conversely, knockdown of HIF-1alpha by RNA interference prevents hypoxia-induced resistance to 5FU. PMX290, a thioredoxin-1 inhibitor, significantly inhibits HIF-1 activity and concomitantly sensitises hypoxic cells to 5FU. These results were confirmed in HCT116 cells grown as three-dimensional spheroids, a model that more closely reproduces the hypoxic environment of solid tumours.

Fluoroquinolone–macrolide combination therapy for chronic bacterial prostatitis: retrospective analysis of pathogen eradication rates, inflammatory findings and sexual dysfunction

We previously demonstrated the safety and efficacy of fluoroquinolone-macrolide combination therapy in category II chronic bacterial prostatitis (CBP). The aim of this study is to retrospectively compare the microbiological and clinical findings of two treatment schemes for CBP based on the combination of azithromycin (500 mg, thrice-weekly) with a once-daily 500- or 750-mg dose of ciprofloxacin (Cipro-500 or Cipro-750 cohort, respectively). Combined administration of azithromycin (1500 mg week(-1)) with ciprofloxacin at the rate of 750 mg day(-1) for 4 weeks rather than at 500 mg day(-1) for 6 weeks increased the eradication rates from 62.35% to 77.32% and the total bacteriological success from 71.76% to 85.57%. A significant decrease in pain and voiding signs/symptoms and a significant reduction in inflammatory leukocyte counts and serum prostate-specific antigen (PSA) were sustained throughout an 18-month follow-up period in both groups. Ejaculatory pain, haemospermia and premature ejaculation were significantly attenuated on microbiological eradication in both groups, but the latter subsided more promptly in the Cipro-750 cohort. In total, 59 Cipro-750 patients showed mild-to-severe erectile dysfunction (ED) at baseline, while 22 patients had no ED on microbiological eradication and throughout the follow-up period. In conclusion fluoroquinolone-macrolide therapy resulted in pathogen eradication and CBP symptom attenuation, including pain, voiding disturbances and sexual dysfunction. A once-daily 750-mg dose of ciprofloxacin for 4 weeks showed enhanced eradication rates and lower inflammatory white blood cell counts compared to the 500-mg dose for 6 weeks. Our results are open to further prospective validation.

Topographic and quantitative relationship between prostate inflammation, proliferative inflammatory atrophy and low-grade prostate intraepithelial neoplasia: A biopsy study in chronic prostatitis patients

Inflammatory processes are important components in the pathogenesis of many human cancers. According to the ‘injury and regeneration’ model for prostate carcinogenesis, injury caused by pathogens or pro-inflammatory cytotoxic agents would trigger proliferation of prostatic glandular cells, leading to the appearance of epithelial lesions named ‘Proliferative Inflammatory Atrophy’ (PIA). Inflammatory cells infiltrating the prostate would release genotoxic reactive oxygen species, leading atrophic cells to neoplastic progression. The hypothesis pointing to PIA as risk-lesion for prostate cancer has been extensively investigated at the cellular and molecular levels, but few morphological data are available linking PIA or prostatic intraepithelial neoplasia (PIN) to inflammation or clinical prostatitis. We investigated at the morphological level 1367 prostate biopsies from 98 patients with a recent history of chronic prostatitis, and 32 patients with biopsies positive for carcinoma. Our results show that i) PIA is found more frequently in biopsy cores containing a severe or moderate inflammatory focus, compared to NON-PIA lesions (partial or cystic atrophy); ii) the PIA lesion post-atrophic hyperplasia is more frequently found in tissues showing mild or no inflammation; iii) the extent of PIA per patient correlates with the burden of moderate or severe inflammation, whereas NON-PIA lesions do not; iv) low-grade PIN is in over 90% of cases emerging from normal, non-atrophic glands and is more frequently found in biopsy cores with absent or mild inflammatory burden; v) the inverse relationship between the prevalence of low-grade PIN and the extent of PIA lesions per patient is described by a power law function, suggesting the low likelihood of the concomitant presence of these lesions in the same tissue; vi) NON-PIA lesions correlate inversely with neoplasia in patients with prostate cancer; vii) the total scores of the NIH-CPSI questionnaire correlate with both PIA and inflammation burdens at diagnosis of prostatitis but not after pharmacological intervention. These results point to a positive association between tissue inflammation, clinical prostatitis and the putative cancer risk-lesion PIA, but do not support a model whereby low-grade PIN would arise from PIA.

p21Waf1/Cip1 and p53 are downstream effectors of protein kinase C delta in tumor suppression and differentiation in human colon cancer cells

We have previously demonstrated that the delta isoform of protein kinase C (PKCδ) is importantly involved in cell growth inhibition and tumor suppression in colon cancer cells. To investigate further the activity and mechanism of action of PKCδ, we have retrovirally transduced a PKCδ cDNA in HCT116 human colon cancer cells. PKCδ‐overexpressing cells (HCT116/PKCδ) were growth‐inhibited, showed marked morphologic changes and underwent multinucleation and phenotypic changes characteristic of mitotic catastrophe. Compared to controls, HCT116/PKCδ cells showed a highly attenuated tumorigenic profile and poor anchorage‐independent growth. In addition, transfected cells established junction‐coordinated intercellular communications, expressed cell surface microvilli and overexpressed the colon differentiation marker alkaline phosphatase. HCT116/PKCδ cells also produced the 89 kDa, carboxy‐terminal catalytic domain of PARP. In HCT116/PKCδ cells, p21Waf1/Cip1 and p53 were transiently upregulated for 48 hr after PKCδ transduction. In a p21 null subline of HCT116 cells (HCT116/p21null), overexpression of PKCδ did not affect tumorigenicity or differentiation, indicating that p21 is essential for the antitumorigenic activity of PKCδ. Similarly, overexpression of PKCδ caused no significant phenotypic changes in HCT116/E6 cells, an HCT116 subline in which the p53 protein is downregulated by the human papillomavirus E6 gene product. We conclude that overexpression of PKCδ in human colon cancer cells induces multiple antineoplastic effects that depend on the activities of p21Waf1/Cip1 and p53.

Multimodal therapy for category III chronic prostatitis/chronic pelvic pain syndrome in UPOINTS phenotyped patients.

The complex network of etiological factors, signals and tissue responses involved in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) cannot be successfully targeted by a single therapeutic agent. Multimodal approaches to the therapy of CP/CPPS have been and are currently being tested, as in the frame of complex diagnostic-therapeutic phenotypic approaches such as the urinary, psychosocial, organ-specific, infection, neurological and muscle tenderness (UPOINTS) system. In this study, the effect of combination therapy on 914 patients diagnosed, phenotyped and treated in a single specialized prostatitis clinic was analyzed. Patients received α-blockers, Serenoa repens (S. repens) extracts combined or not with supplements (lycopene and selenium) and, in the presence of documented or highly suspected infection, antibacterial agents. Combination treatment induced marked and significant improvements of National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) prostatitis symptom scores, International Index of Erectile Function (IIEF) sexual dysfunction scores, urinary peak flow rates and bladder voiding efficiency. These improvements, assessed after a 6-month course of therapy, were sustained throughout a follow-up period of 18 months. A clinically appreciable reduction of ≥6 points of the total NIH-CPSI score was achieved in 77.5% of patients subjected to combination therapy for a period of 6 months. When the patients were divided in two cohorts, depending on the diagnosis of CP/CPPS [inflammatory (IIIa) vs. non-inflammatory (IIIb) subtypes], significant improvements of all signs and symptoms of the syndrome were observed in both cohorts at the end of therapy. Intergroup comparison showed that patients affected by the IIIa sub-category of CP/CPPS showed more severe signs and symptoms (NIH-CPSI total, pain and quality of life impact scores, and Qmax) at baseline when compared with IIIb patients. However, the improvement of symptoms after therapy was significantly more pronounced in IIIa patients when compared with IIIb patients. In contrast to current opinion, the evidence emerging from the present investigation suggests that the inflammatory and non-inflammatory sub-categories of CP/CPPS may represent two distinct pathological conditions or, alternatively, two different stages of the same condition. In conclusion, a simple protocol based on α-blockers, S. repens extracts and supplements and antibacterial agents, targeting the urinary, organ specific and infection domains of UPOINTS, may induce a clinically appreciable improvement of the signs and symptoms of CP/CPPS in a considerable percentage of patients. In patients not responding sufficiently to such therapy, second-line agents (antidepressants, anxiolytics, muscle relaxants, 5-phosphodiesterase inhibitors and others) may be administered in order to achieve a satisfactory therapeutic response.