Gian Paolo Trentini

Dystroglycan Expression Is Frequently Reduced in Human Breast and Colon Cancers and Is Associated with Tumor Progression

Dystroglycan (DG) is an adhesion molecule responsible for crucial interactions between extracellular matrix and cytoplasmic compartment. It is formed by two subunits, alpha-DG (extracellular) and beta-DG (transmembrane), that bind to laminin in the matrix and dystrophin in the cytoskeleton, respectively. In this study we evaluated by Western blot analysis the expression of DG in a series of human cancer cell lines of various histogenetic origin and in a series of human primary colon and breast cancers. Decreased expression of DG was observed in most of the cell lines and in both types of tumors and correlated with higher tumor grade and stage. Analysis of the mRNA levels suggested that expression of DG protein is likely regulated at a posttranscriptional level. Evaluation of alpha-DG expression by immunostaining in a series of archival cases of primary breast carcinomas confirmed that alpha-DG expression is lost in a significant fraction of tumors (66%). Loss of DG staining correlated with higher tumor stage (P = 0.022), positivity for p53 (P = 0.033), and high proliferation index (P = 0.045). A significant correlation was also observed between loss of alpha-DG and overall survival (P = 0.013 by log-rank test) in an univariate analysis. These data indicate that DG expression is frequently lost in human malignancies and suggest that this glycoprotein might play an important role in human tumor development and progression.

Cyclin D1 expression in papillary superficial bladder cancer: its association with other cell cycle-associated proteins, cell proliferation and clinical outcome.

Cyclin D1 contributes to regulate G1 progression by forming a complex with different cyclin‐dependent kinases. It has oncogenic properties and is frequently overexpressed in several human tumor types. In our study, expression of cyclin D1 and Ki67, a proliferation marker, was evaluated by immunohistochemistry in human papillary superficial (pTa‐pT1) bladder cancers and was correlated with p27Kip1, p21Waf1 and c‐erbB‐2 expression, with p53 gene status and protein expression, ploidy and cancer progression. Cyclin D1 expression was neither associated with tumor stage nor with tumor grade but high cyclin D1 expression (≥25% positive nuclei) was significantly associated with p53 gene mutation (p = 0.012), low p21Waf1 (p = 0.015) and high p27Kip1 (p = 0.016) protein expression. Ki67 expression was not associated with tumor stage but a high proliferation index (≥10% positive nuclei) was significantly associated with high tumor grade (p = 0.001) and with DNA aneuploidy (p = 0.005). There was no significant difference in proliferative activity between high and low cyclin D1 expressor tumors. Patients whose tumors showed high expression of cyclin D1 displayed a significantly longer disease‐free survival (p < 0.001 by log‐rank test). Increased Ki67 expression was significantly associated with shorter disease‐free survival (p = 0.003). Both cyclin D1 (p = 0.027; RR = 1.898) and Ki67 (p = 0.047; RR = 1.932) protein expressions were independent predictors of reduced disease‐free survival on a multivariate analysis that also included p27Kip1 expression and tumor stage. The simultaneous presence of low cyclin D1, low p27Kip1 and high Ki67 expression defined a “high‐risk” group of patients who displayed a significantly increased risk of recurrence (p < 0.0001). These results suggest that evaluation of cell cycle‐associated markers can help to identify high‐risk patients and may affect the management of patients with papillary superficial bladder cancer.

Mismatch repair proteins expression and microsatellite instability in skin lesions with sebaceous differentiation : A study in different clinical subgroups with and without extracutaneous cancer

Muir-Torre syndrome (MTS) is defined as the association of a sebaceous tumor or keratoacanthoma and an extracutaneous neoplasm, mainly from the gastrointestinal or genitourinary tracts. MTS is related to hereditary non-polyposis colorectal cancer (HNPCC), a syndrome with germline mutations in the mismatch repair (MMR) gene(s), leading to microsatellite instability (MSI). In this study, using immunohistochemistry and a microsatellite instability assay, we analyzed the incidence of MMR gene abnormalities in 79 sebaceous lesions from 70 patients, 26 of whom also had an extracutaneous visceral neoplasm. We were unable to investigate the family histories of our patients regarding other tumors in order to assess which of our cases met the Amsterdam criteria. Defective MMR protein expression (MMR-) was found in 18/70 (25.7%) patients, with an identical distribution between those having an isolated skin tumor (11/44, 25.0%) and those with an extracutaneous cancer (7/26, 25.4%). In the sporadic group, MMR negative lesions were significantly more frequent in extrafacial areas (P = 0.03). High concordance was found between MMR expression in sebaceous lesions and the extracutaneous neoplasm in the same patient (20/23, 86.9%), as well as between MMR expression and microsatellite status (18/20, 90%). In conclusion, this study confirms the value of immunohistochemistry to identify MMR defective tumors. However, since only a minority of sebaceous neoplasms in patients who also have an extracutaneous cancer display MMR defects, these techniques are of limited value for the identification of “clinically defined” MTS.

Superficial papillary urothelial carcinomas in young and elderly patients: a comparative study

To compare the clinicopathological and immunohistochemical findings of superficial papillary transitional cell carcinomas in ‘young’ and ‘elderly’ patients, as the natural history and prognosis of bladder tumours in young patients remains a matter of debate.

Estrogens Modulate the Circadian Rhythm of Hypothalamic Beta-Endorphin Contents in Female Rats

The aim of the present study was to evaluate the changes in the diurnal rhythm of the hypothalamic beta-endorphin (beta-EP) contents in female rats as a function of circulating estrogens. With this purpose we evaluated the diurnal hypothalamic beta-EP changes (1) during the estrous cycle, and (2) in ovariectomized rats with and without acute and chronic estrogen replacement. Ovariectomized rats were treated either acutely with 10 micrograms of estradiol benzoate (EB) or chronically with 2 micrograms/day of EB for 15 days. beta-EP concentrations were measured in acid extracts of medial basal hypothalamus by a specific radioimmunoassay. During the estrous cycle, hypothalamic beta-EP concentrations showed a significant nocturnal increase, with no difference between the 4 days of the cycle. On the day of estrus, beta-EP concentrations between 12.00 and 18.00 h resulted significantly lower than in the other days of the cycle. After ovariectomy, the night-related changes in hypothalamic beta-EP disappeared. The acute administration of EB induced a significant increase in hypothalamic beta-EP after 21 h (18.00 h). On the other hand, the chronic replacement restored the nocturnal peak of hypothalamic beta-EP (18.00, 21.00, 24.00 h). The present data emphasize the role of central beta-EP in regulating the reproductive functions. Moreover, the effect of estrogen in modulating the circadian changes in hypothalamic beta-EP supports the important role of estrogens in brain function.

Detection of human papillomavirus in extragenital Bowen's disease using in situ hybridization and polymerase chain reaction.

Extragenital Bowens disease (EBD) has rarely been studied for the presence of human papillomaviruses (HPVs). Twenty consecutive patients with EBD were investigated for the presence of HPVs using in situ hybridization with a generic probe that can detect HPV DNA types 6, 11, 16, 18, 30, 31, 33, 35, 45, 51, and 52 and specific probes for HPV DNA types 6/11, 16/18, and 31/33/35. All cases were tested with the polymerase chain reaction (PCR) technique employing the L1 consensus primer pair, MY11 (primer for the positive strand) and MY9 (primer for the negative strand) complementary to genital and dermal HPV types. Seven Caucasian patients, five males and two females, with an average age of 70.4 years, showed positive in situ hybridization (ISH) for HPV DNA. The positivity varied from 5 to 40% of neoplastic cells. Three of seven of the ISH DNA-positive cases showed a positive PCR for DNA HPVs. The role of HPVs in human tumors is not fully understood since oncogenic types of HPVs have been found in normal tissue and the actions of cofactors have been postulated. Bowens disease usually occurs in elderly people in whom the efficiency of the immune systems may be compromised. The association between HPV infection and low efficiency of the immune response may be responsible for HPV-related Bowens disease in elderly people.

Detection of human papillomavirus DNA in urinary bladder carcinoma by in situ hybridisation.

AIMS: To investigate the sensitivity of an in situ hybridisation system to detect human papillomavirus (HPV) infection in transitional cell bladder cancer and to evaluate the advantages of analysing multiple biopsies; to examine the correlation between HPV tumour infection detected by in situ hybridisation and the presence of serum anti-HPV antibodies detected by enzyme linked immunosorbent assay (ELISA); and to relate the presence of viral infection to grade, stage, and follow up in cases of bladder cancer. METHODS: The in situ hybridisation technique was used with broad spectrum and type specific (6/11, 16/18, 31/33/35) probes against HPV DNA in formalin fixed, paraffin embedded tissues from 43 cases of bladder cancer. The results were analysed for the presence and type of papillomavirus and correlated with clinicopathological variables. RESULTS: The presence of HPV DNA was identified by the in situ hybridisation technique in 17 of 43 cases of bladder cancer; 12 of these were serum antibody positive and 10 had had multiple biopsies. Fifteen of the cases that were negative for HPV DNA by in situ hybridisation had positive serum serology when tested by ELISA. In 14 cases, the HPV was either types 16/18 or types 31/33/35, both of which carry high oncogenic risk. The stage (p < 0.05) and grade (NS) of the tumour and the outcome on follow up (p < 0.05) were correlated with the presence of HPV infection. CONCLUSIONS: ELISA is not useful in identifying patients with HPV positive bladder cancer, but the use of several probes and multiple biopsies increases the detection rate of HPV in neoplastic tissues. The association between tumour virus infection and high grade/high stage tumours and worse outcome suggests that HPV infection of neoplastic tissue has a negative effect on the behaviour and evolution of transitional cell bladder carcinoma.

Effect of chronic intermittent stress on rat pregnancy and postnatal development

The present study evaluates the effect of chronic intermittent cold-swimming stress on body weight gain of pregnant rats and subsequent development of the offsprings after birth, till peripubertal stage. When stress was administered during the first half (1-11 days) of gestation, weight gain of pregnant rats was significantly lower at the 9th and 11th days (P < 0.05 vs. control, respectively). No differences of weight gain in comparison with control rats were found at term gestation in pregnant rats exposed to stress continuously. Similarly, stress administered, starting from 12th day till term gestation, had no effect on weight gain. Even though weight gain of pregnant rats during the second half of pregnancy in group stress 1-11 was restored to normal values, a high mortality rate of neonates 1, 2 and 3 weeks after birth was found in this group (P < 0.02, 0.01 and 0.001 vs. controls). There was no significant difference between stressed and control groups with respect to the number or body weight of litters, as well as weight gain of neonates during the first 21 days of life. In addition, in offsprings from all stressed groups, a high number of small for date animals was found after 14 days of life, and 74.4% of these small for date animals died during the peripubertal period. The present data demonstrate that the exposure to stress in utero may induce damaging effects on postnatal development.

Effect of different chronic intermittent stressors and acetyl-l-carnitine on hypothalamic beta-endorphin and GnRH and on plasma testosterone levels in male rats.

Chronic stress affects the reproductive function by modifying the neuroendocrine homeostasis. The aim of the present study was to clarify the neuroendocrine and the gonadal changes following chronic intermittent stress in male rats and the action of a neuroactive drug, acetyl-l-carnitine (ALC). The effect of two different stressors, cold water swimming or ether, on central beta-endorphin (beta-EP) and GnRH contents, and on plasma testosterone levels was investigated. In addition, the response to an acute stress in chronically stressed rats, treated or untreated with ALC (10 mg/day/rat p.o.), was evaluated. The stressors were applied twice a day for 10 days, and rats were killed before, during and after the last stress session. Mediobasal hypothalamus (MBH) beta-EP and GnRH contents, and plasma testosterone levels were evaluated by radioimmunoassay. The following results were obtained: (1) both chronic swimming and ether stress caused a decrease in hypothalamic beta-EP contents; (2) MBH GnRH contents increased after chronic swimming stress but not after ether stress; (3) chronic swimming stress induced a twofold decrease in plasma testosterone levels, while no changes were observed after ether stress; (4) the treatment with ALC prevented the decrease in plasma testosterone levels after chronic swimming stress, and (5) acute stress in chronically stressed animals caused an increase in MBH-beta-EP. The present data showed that chronic swimming stress reduces the reproductive capacity and impairs the capacity to respond to the acute stress and that ALC modulates the hormonal changes to physical stress and prevents the antireproductive effect of chronic cold swimming.

Melatonin treatment delays reproductive aging of female rat via the opiatergic system.

In female rat age-related reproductive decline is accompanied by progressive impairment of the neuroendocrine mechanisms that regulate LH secretion. The biosynthetic activity of the pineal gland is markedly depressed and the nocturnal secretion of melatonin decreases significantly. The aim of the present study was to evaluate whether the nocturnal administration of melatonin via the drinking water (0.4 micrograms/ml) throughout the course of aging from 14 to 24 months of age could (1) influence the age-related changes that occur in basal serum levels of LH and in the LH response to GnRH or to naloxone stimulation at 16, 18 and 20 months of age, and (2) delay the onset of the postreproductive constant estrous-anovulatory state as evaluated by the daily recording of vaginal smears and by occurrence of polyfollicular ovaries at 24 months of age. Our results demonstrate that melatonin replacement delays the increase in LH serum levels and the decrease in LH response to GnRH that occur in 18-month-old control animals. Furthermore, they show that melatonin treatment prevents the loss of LH response to naloxone manifested in control rats between 16 and 20 months of age. Melatonin also appears to prevent the progressive increase in the monthly occurrence of estrus phases as well as to decrease the number of rats with polyfollicular ovaries at 24 months of age in comparison to control animals. These results suggest that the age-related decrease in circulating melatonin during the night may contribute to the reproductive decline of aging, and that this effect may involve the central opioid system.