p21WAF1/CIP1 protein expression in primary ovarian cancer.

p21WAF1/CIP1 protein is a cyclin-dependent kinase inhibitor, able to prevent the CDK2/cyclin E induced retinoblastoma protein (pRB) phosphorylation, thus inhibiting cell cycle progression at G1 phase. p21WAF1/CIP1 protein levels were examined in a series of 102 ovarian tissue samples including normal ovary, primary ovarian tumors, omental metastasis, recurrent disease and residual tumor after chemotherapy exposure, by Western blot analysis. The association of p21WAF1/CIP1 status with clinicopathological parameters and clinical outcome was also investigated. p21WAF1/CIP1 protein was detectable in 76 out of 102 (74%) ovarian tissue samples. We observed a significant trend of p21 levels to gradually increase from normal ovarian tissues (median 0 a.u.) through primary ovarian cancers (median 0.19 a.u.), omental metastases (median 0.33 a.u.) and recurrence of disease (median 0.44 a.u.) (p=0.015). In the group of stage III-IV ovarian cancer patients, p21-positive cases showed a more favourable prognosis with respect to p21-negative cases: the 3-year time to progression (TTP) rate was 58% for p21-positive compared with 33% of p21-negative cases (p=0.036). In conclusion, p21WAF1/CIP1 expression levels seem to be correlated with tumor status at the time of diagnosis and can predict TTP in a selected group of patients.

Bcl-2, Bax, Bcl-x(L) and Bcl-x(S) expression in neoplastic and normal endometrium.

The two anti-apoptotic proteins Bcl-2 and Bcl-x(L), and the two pro-apoptotic proteins Bax and Bcl-x(S) were measured by Western blotting in 51 neoplastic and 8 normal endometrial samples. The corresponding mRNA levels were analyzed by semiquantitative reverse transcriptase-polymerase chain reaction in a subgroup of 19 endometrial carcinomas. Neoplastic tissues had higher amounts of Bcl-2 protein than normal tissues (p < 0.051). Bcl-x(L) followed the same trend since its levels were higher in tumor than in normal samples (p < 0.048). Interestingly, Bcl-2 and Bcl-x(L) protein content showed a trend towards an inverse correlation (r = 0.27, p < 0.052). mRNA and protein levels directly correlated only with Bcl-2 (r = 0.63, p < 0.0032). Despite the fact that the amounts of Bcl-2, Bax and Bcl-x(L) proteins in the neoplastic population were not significantly differently distributed according to the clinicopathological features of the patients, the differences observed between normal and neoplastic samples suggest that these proteins may play a role in endometrial carcinoma: long-term follow-up studies will be required to confirm this hypothesis.

Expression of bcl-2, bax-xL, and bcl-xS in endometrial and cervical tissues.

We assessed the protein expression levels of bcl-2, bax, bcl-xL, and bcl-xS in a group of 51 endometrial cancers and 8 normal samples as well as in 59 cervical neoplasms and in 15 normal cervical tissues. Neoplastic endometria (median, 1.30 absorbance units [AU]; range, 0.13-7.26 AU) had slightly higher bcl-2 levels than did normal tissue (median, 0.83 AU; range, 0.29-1.90 AU; P < .068), whereas bcl-2 was lower in neoplastic (median, 3.59 AU; range, 0.13-19.86 AU) than in normal cervical samples (median, 8.45 AU; range, 2.09-15.04 AU; P < .010). Bcl-xL levels were higher in endometrial carcinoma (median, 1.23 AU; range, 0.03(4.29 AU) than in normal tissues (median, 0.56 AU; range, 0.46-1.48 AU; P < .048), whereas no significant difference was observed in cervical tissues. Bax levels did not show any variation in either system. The protein bcl-xS was marginally detectable in only a few samples. In endometrial carcinoma, bcl-2 and bcl-xL levels were correlated inversely (r = -0.27; P < .054), whereas in cervical cancer, they were correlated directly (r = +0.40; P < .002). The different expression patterns of bcl-2 family members in endometrial and cervical tissues confirm the hypothesis of a strictly tissue-specific regulation of these proteins.

p53 overexpression is associated with cytoreduction and response to chemotherapy in ovarian cancer.

The aim of this study was to assess the association of p53 status with primary cytoreduction, response to chemotherapy and outcome in stage III-IV primary ovarian cancer patients. Immunohistochemical analysis of p53 was performed on formalin-fixed, paraffin-embedded specimens from 168 primary ovarian carcinomas by using the DO-7 monoclonal antibody. p53 nuclear positivity was found in 84 out of 162 (52%) malignant tumours. A higher percentage of p53 nuclear positivity was observed in patients with advanced stage of disease than in stage I-II (57% vs 23% respectively; P = 0.0022) and in poorly differentiated versus well/moderately differentiated tumours (59% vs 32% respectively; P = 0.0038). The multivariate analysis aimed to investigate the association of FIGO stage, grade and p53 status with primary cytoreduction in 136 stage III-IV patients showed that stage IV disease may influence the possibility to perform primary cytoreduction in ovarian cancer patients. p53-positivity also maintained a trend to be associated with poor chance of cytoreduction. In patients who underwent pathologic assessment of response, cases who did not respond to chemotherapy were much more frequently p53-positive than p53-negative (86% vs 14% respectively; P = 0.012). Moreover, patients with stage III disease and < 2-cm residual tumour were more likely to respond to treatment. In multivariate analysis, FIGO stage and p53 expression were independently correlated with pathologic response to chemotherapy. Time to progression and survival rates were shown not to be different in p53-positive versus p53-negative patients.

bcl-2, bax, bcl-XL, and bcl-XS expression in normal and neoplastic ovarian tissues.

The bcl-2 family of proteins includes some important regulators of apoptosis. Among these, bcl-2 and bcl-xL prevent cells from entering apoptosis, whereas bax and bcl-xS can induce cell death. Alterations in the control of this process can lead to a decrease in cell death, thus contributing to neoplastic growth. Diminished susceptibility to chemotherapy has also been attributed, in in vitro systems, to alterations in the levels of bcl-2, bax, or bcl-x. We analyzed the expression of bcl-2, bax, bcl-xL, and bcl-xS in normal and neoplastic ovarian tissues by reverse transcriptase-PCR and Western blotting. The RNA and protein levels were significantly correlated for all genes. Interestingly, the levels of these genes in normal and neoplastic tissues were significantly different: bcl-2 was higher in normal tissue (P < 0.002), whereas bax and bcl-xL were higher in carcinoma (P < 0.018 and P < 0.030, respectively). bcl-xS was present at low levels in 83% of neoplastic samples and was undetectable in normal tissue. Reverse transcriptase-PCR analysis of 74 tumors showed no major correlation with clinicopathological parameters or with response to chemotherapy. Only bax and bcl-xL were correlated with progesterone receptor levels (n = 29, r = +0.44, P < 0.0189, and r = -0.40, P < 0.035, respectively). No correlation was found with estrogen receptor levels or with p53 immunostaining. Our data indicate that the regulation of the bcl-2 family of proteins differs between normal and neoplastic ovarian tissues. Moreover, the modulation of these genes in ovarian carcinoma is different compared to other tissues; therefore, tissue specificity is very important in regulation of the bcl-2 family of proteins.

Risk factors for micro- and macroangiopathic complications in type 2 diabetes: lack of association with acetylator phenotype, chlorpropamide alcohol flush and ABO and Rh blood groups.

Genetic markers would be useful to study the transmission of type 2 diabetes and to identify patients with enhanced risk of development of late diabetic complications. The aim of this study was to evaluate the influence of selected possible genetic markers on the development of diabetic complications. One hundred and eighty patients with type 2 diabetes (79 males, 101 females) were therefore studied with respect to ABO and Rh blood grouping and chlorpropamide alcohol flush (CPAF) and acetylator phenotype status, in addition to life style (smoking, dietary, alcohol and drug taking habits) and metabolic indexes (HbA1, M-value, serum cholesterol, serum triglycerides), with regard to late complications coronary heart disease (CHD), arterial hypertension (AH), peripheral vascular disorders (PVD), retinopathy and nephropathy. None of the genetic markers considered appeared to be associated with diabetic complications. Multiple logistic analysis identified different risk-factors for each complication: AH and age for CHD; hyperlipidaemia for AH; age of patients for PVD; duration of diabetes for retinopathy; AH for nephropathy. It is concluded that the possible genetic markers evaluated in this study do not identify a higher or lower risk for late complications. On the contrary, most of the risk factors identified support previous studies. Active correction of these risk-factors might improve the overall prognosis of patients with type 2 diabetes.

Puerperal lactation, gonadotropin release and estradiol release: effects of metergoline and bromocriptine.

The exact cause of amenorrhea during the puerperium is still a matter of debate. PRL might inhibit primarily the release of FSH and LH or their stimulating effects on the ovary. In the study presented here, 28 healthy women were investigated, 13 of them lactating puerperae. In the other 15, lactation was prevented by drugs (metergoline in 9, bromocriptine in 6). The women's serum PRL, FSH, LH, beta-HCG and 17 beta-estradiol as well as their FSH and LH response to LHRH (100 micrograms i.v.) were tested 1, 3, 7 and 14 days after vaginal delivery. Serum PRL levels remained elevated in the lactating puerperae and dropped in the puerperae treated with metergoline or bromocriptine. The pattern of FSH, LH and beta-HCG levels as well as the FSH and LH response to LHRH were superimposable in lactating and in nonlactating women. 17 beta-estradiol levels dropped in all puerperae from day 1 to 7, but rose from day 7 to 14 only in the puerperae treated with metergoline or bromocriptine and not in the lactating women. These data indicate that PRL directly affects the ovarian response to FSH and LH, whereas the release of FSH and LH remains unaffected. A stimulatory effect of metergoline and bromocriptine on the ovarian steroidogenesis cannot be excluded.

Genetic and metabolic risk factors for the development of late complications in type I (insulin-dependent) diabetes.

The genetic background seems to be involved in the development of type I diabetes and it might also be involved in the development of diabetic complications, but studies carried out so far have yielded conflicting results. The aim of this study was to evaluate the influence of some genetic markers and metabolic factors in the development of late diabetic complications. One hundred and twenty-seven patients (69 males, 58 females) with type I diabetes were evaluated for ABO and Rh blood groups, chlorpropamide alcohol flush (CPAF) and acetylator phenotype (AP) as well as for life-habits (smoking, alcohol use, diet and drug compliance), metabolic indexes (M-value, HbA1, cholesterol and triglyceride levels) and late complications of diabetes [coronary heart disease (CHD), arterial hypertension (AH), retinopathy and nephropathy]. Diabetic patients were more frequently fast acetylators and CPAF positive than controls and CPAF was more frequent among females than among males. None of the genetic markers used in this study appeared as a risk factor for the development of diabetic complications. At multiple logistic analysis different risk factors appeared for each microangiopathic complication. For retinopathy: female sex, duration of disease and triglyceride levels; for nephropathy: male sex, cholesterol levels and hypertension. These risk factors have already been recognized in previous studies, while the genetic markers evaluated in our study do not identify a greater or smaller risk for the development of late complications.

Association between chlorpropamide-alcohol flushing and fast acetylator phenotype in type I and type II diabetes.

Different prevalences of chlorpropamide alcohol flushing (CPAF) have been reported by different authors in either type I or type II diabetics or in normal subjects and this could be due to different methodological approaches or to different criteria of evaluation of CPAF. Previous studies in small series of patients have also suggested the existence of an association between type I diabetes and the fast acetylator phenotype (AP). The first aim of this study was to find reliable criteria for the assessment of CPAF. The second was to evaluate the prevalence of CPAF and of AP in a large series of type I and type II diabetics; and the third was to evaluate possible associations of CPAF and AP. AP and CPAF were evaluated separately in 256 diabetics (110 with type I and 146 with type II diabetes) and in 183 diabetics (74 with type I and 109 with type II diabetes), respectively. In 156 of these subjects, the two markers were evaluated together. The occurrence of CPAF was studied by subjective and objective assessment and by thermographic recording; CPAF was quantified by the differential value of skin temperature increase [delta T(C-P)] and by the value of differential speed of ascent, expressed in angle-degrees [delta a(C-P)], after treatment with placebo and with chlorpropamide. The fast AP was more frequent in type I than in type II diabetics, was not related to family history of diabetes, sex of the patients, age at onset and duration of diabetes or metabolic control. The most reliable assessment of CPAF was represented by thermographic recording of speed of ascent of skin temperature. CPAF was more frequent in females than in males, more frequent in diabetics than in healthy controls, similarly frequent in type I and in type II diabetes and showed no relationship with family history of diabetes, age at onset, duration of diabetes or metabolic control. An association between fast AP and CPAF was found in type II, but not in type I diabetics: fast acetylators were more frequently CPAF-positive, while slow acetylators were more frequently CPAF-negative. In addition, a linear relationship was found between rate of acetylation and speed of ascent of facial skin temperature after chlorpropamide and alcohol in type II diabetics, not in type I diabetics. The meaning of this association is not clear and deserves further investigations.

Effect of metergoline on prolactin, follicle stimulating hormone, luteinizing hormone and thyroid stimulating hormone response to TRH and LHRH in normal men and women.

Metergoline, a prolactin (PRL)-lowering drug with an antiserotoninergic activity, is known to restore menstruations and fertility in hyper-PRL patients even when PRL levels are not normalized. This suggests that metergoline might also affect gonadotropins release. In a double-blind cross-over study in 8 normal males, repeated administration of metergoline enhanced the LH response to LHRH and reduced the PRL response to TRH; for females, three different tests were performed on days 5, 8 and 21 of two different menstrual cycles, each test being preceded by metergoline or by placebo administration. Metergoline always reduced the PRL response to TRH; on the 5th day, metergoline reduced the FSH response to LHRH and on the 21st day enhanced the LH response to LHRH. Basal levels of LH, FSH, T3, T4, Testosterone, 17 beta-estradiol and progesterone as well as the FSH response to LHRH (in males) and the TSH response to TRH (in both males and females) were not modified by metergoline. The data suggest that tests with TRH and LHRH can yield different results when performed during metergoline administration and that metergoline, acting through an unknown mechanism, can modify gonadotropins release.

The fast acetylator phenotype in diabetes mellitus: abnormal prevalence and association with the ABO blood groups.

The acetylator phenotype and ABO blood groups were evaluated in 55 normal subjects and in 156 diabetic patients [61 with Type 1 (insulin-dependent) diabetes and 95 with Type 2 (non-insulin-dependent) diabetes]. The prevalence of fast acetylators was significantly higher in the Type 1 diabetic patients (53%) than in the control subjects (29%). In the Type 2 diabetic patients the prevalence was 39%, and thus not significantly different from the control or Type 1 diabetic groups. In the Type 2 diabetic patients, but not in the control or in the Type 1 diabetic subjects, an association between the fast acetylator phenotype and the B blood group was found.

Characterization of the chlorpropamide-alcohol-flush in patients with type 1 and type 2 diabetes.

The aim of the present paper was to evaluate the prevalence of the chlorpropamide-alcohol-flush (CPAF) in patients with type 2 and with type 1 diabetes. Ninety-seven patients with type 2 diabetes and 33 with type 1 diabetes drank 40 ml vermouth 12 h after placebo and again 12 h after 1 tablet of chlorpropamide (250 mg) or 12 h after the last of repeated administrations of chlorpropamide (250 mg b.i.d. for 2 days). Skin temperature was recorded in all patients by a thermocouple probe connected to the left cheek. In 47 patients serum concentrations of chlorpropamide and of its metabolite CBSU were also determined. The prevalence of CPAF was similar in type 1 and type 2 diabetes, was greater in women than in men, and was significantly greater after repeated administrations than after one single administration of chlorpropamide. The increase of skin temperature during a 30-min period was significantly higher in patients with CPAF than in patients without CPAF. Serum concentrations of chlorpropamide and of its metabolite CBSU were more elevated after 4 than after 1 tablet of chlorpropamide, but were not significantly different in patients with and without CPAF. These data indicate that both genetic factors and the amount of chlorpropamide used affect the appearance of CPAF. To assess the possible role of serotonin and of dopamine in the CPAF, some patients with CPAF were tested again after treatment with metergoline, an antiserotonin agent, or with bromocriptine, a dopamine-agonist. Neither drug influenced the CPAF, indicating that the two neurotransmitters are not involved in the CPAF.

The effect of histamine and H1 and H2 receptors on prolactin and luteinizing hormone release in humans: sex differences and the role of stress.

The present experiments were performed to investigate the possible role of histamine and its receptors, H1 and H2, in the control of PRL and LH release in normal adult humans of both sexes. Histamine infusion (200 microgram, iv, in 15 min) induced PRL and LH release in men; in women, histamine inhibited LH release without affecting PRL release. Two H1 antagonists, dexchlorpheniramine (10 mg, iv) and promethazine (50 mg, im), reduced PRL release in both sexes, stimulated LH release in men, and inhibited LH release in women. Cimetidine, an H2 antagonist (400 mg, iv), elicited PRL release in both sexes, more consistently in females than in males, and was without effect on LH release in either sex. These data suggest that in humans, the effect of histamine on PRL release is linked to H1 and H2 receptors, which respectively stimulate and inhibit PRL release independently of sex. The effect of histamine on LH release appears to depend on sex and to be mediated only by H1 receptors. To rule out the possibility that the effects of histamine are merely due to a nonspecific stress reaction, we have evaluated PRL and LH release in otherwise normal men and women undergoing surgery for gallstones. Surgery was accompanied by PRL release in both sexes, more evident in women, and by LH release only in men. These results indicate that the effect of histamine on PRL and LH release in humans is linked to sex and H1 and H2 receptors and is not due to stress; further studies are required to clarify the possible mechanism and site of action of histamine in modifying PRL and LH release in humans.

Interaction of dopaminergic and antiserotoninergic drugs in the control of prolactin and LH release in normal women.

Prolactin (Prl) release, both in the experimental animal and in man, is stimulated by serotonin (5HT) and inhibited by dopamine (DA). Data also suggest that LH release in the animal is stimulated by norepinephrine and inhibited by DA. The role of 5HT in the control of LH release is less clear. It would appear to stimulate episodic LH release and to inhibit the LH surge at the pro-oestrus in animals, but the effect of 5HT on LH release has not yet been evaluated in man. In the present paper we have studied the effect of various DA-ergic drugs (DA, iv l-dopa, po l-dopa and bromoergocriptine) and of two anti-5HT drugs, metergoline and methysergide, on Prl and LH release in normal women. DA-ergic drugs lowered plasma Prl and LH levels; anti-5HT drugs, at doses able to lower Prl levels, did not affect basal LH release nor the inhibiting effect of iv l-dopa on LH release. These data indicate that DA inhibits both LH and Prl release in normal women, and that 5HT stimulates Prl release but is not involved in the regulation of LH secretion. The fact that, at variance to all the DA-ergic drugs used, the two anti-5HT drugs did not vary LH release, suggests that metergoline and methysergide are devoid of DA-ergic activity in man, at least at the doses able to inhibit Prl release.