Different effects of interferon-alpha on melanoma cell lines: a study on telomerase reverse transcriptase, telomerase activity and apoptosis.

BACKGROUND: Although the antiproliferative and proapoptotic effects of interferon (IFN)-alpha are widely recognized, its antitumour mechanisms are not completely known. Recent studies indicate that the derepressed expression of the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT), and telomerase activity (TA) are involved in the process of human carcinogenesis. Only a few studies have investigated the effects of IFN-alpha on hTERT and TA, with controversial results. Objectives To study the hTERT mRNA expression, TA and apoptosis in human melanoma cells treated with IFN-alpha. METHODS: Five human melanoma cell lines (Me665/2/21, Me665/2/60, HT-144, SK-Mel-28 and SK-Mel-5) were cultured in standard conditions and treated with 20000 IU mL-1 of human recombinant IFN-alpha-2b. Apoptosis was evaluated as hypodiploid DNA content determined by flow cytometry, caspase-3/7 activity by enzymatic assay, and poly(adenosine diphosphate-ribose) polymerase cleavage by Western blot analysis. IFN-alpha receptor (IFNA-R) and hTERT mRNA expression levels were evaluated by semiquantitative reverse transcription-polymerase chain reaction. TA was evaluated by a polymerase chain reaction-based telomerase repeat amplification protocol assay. RESULTS: Besides a variable degree of cell proliferation inhibition in all cell lines tested, we found different responses, ranging from no significant effects in SK-Mel-28 cells, to a high degree of apoptosis with no hTERT mRNA expression and TA modification in HT-144 cells, and induction of apoptosis, along with decrease in hTERT mRNA expression and TA in Me665/2/21 cells. No induction of apoptosis was observed in SK-Mel-5 and Me665/2/60 cells, although an early decrease in hTERT mRNA expression, and a minor increase of both hTERT mRNA expression and TA were found, respectively. CONCLUSIONS: Our results suggest that the effects of IFN-alpha on hTERT and TA can result from the induction of apoptosis, but they can also occur through a direct modulation of hTERT. We hypothesize that, depending on the cellular context rather than the IFNA-R status of the targeted cells, IFN-alpha can elicit an apoptotic cell death; furthermore, different pathways of apoptosis, not necessarily involving telomerase, can be put into motion.

[Immunotherapy in the management of metastatic renal carcinoma]. / Immunoterapia nel trattamento del carcinoma renale metastatico.

BACKGROUND: Renal cell carcinoma (RCC) is a relatively rare tumor representing 2-3% of all neoplasias. Approximately 30% of patients diagnosed as having RCC present metastases: mean survival ranges between 6 and 10 months, whilst 10-20% present a 2-year survival rate. Treatment of patients with metastatic RCC is a difficult challenge. Hormones and chemotherapy, either alone or associated with surgical resection of the primary lesion have been used. In our experience, IL-2 + IFN-a has been shown to be an encouraging form of treatment. METHODS: During the period between October 1997 and December 1999, 5 patients (3 males, 2 females), mean age 52.5 years, with metastatic RCC, came to our attention. TC revealed a circumscribed area the margins of which were not well defined, in a mid-renal localization, positive inter aorta caval and obturator lymph nodes, but no lung, brain or hepatic involvement. Patients were submitted to nephrectomy and lymphadenectomy followed later by immunotherapy with IFN-alpha (3 Mil twice a week) + low dose IL-2 (1 Mil/m2/12 h): treatment was given for 4 consecutive weeks and then repeated every 3 weeks for 1 year. RESULTS: At 30 months' follow-up all patients showed regression of the disease (100% of cases) and in none of the cases there was evidence of metastases. Only one patient (20%) presented side-effects (nausea, vomiting, slight rise in temperature) all of which disappeared at the end of the first week of treatment. CONCLUSIONS: These results, even if on a limited number of patients and for a limited follow-up period, show that in some patients with metastatic renal cell carcinoma, treatment with IL-2 and IFN-a following nephrectomy and lymphadenectomy should be considered the therapy of choice.

[Stenosis of the new bladder neck, a complication of radical prostatectomy: personal experience]. / Stenosi del neocollo vescicale quale complicanza della prostatectomia radicale.

BACKGROUND: To describe personal experience on stenosis of the new bladder neck, a complication of radical prostatectomy. After a review of the literature, guidelines are proposed to avoid this complication, both from a prophylactic and treatment point of view. METHODS: Between 1992 and 2000, 54 patients were submitted to radical prostatectomy, 18 of whom later presented stenosis in the new bladder neck. Mean age of patients was 65.6 years (range 55-76). According to TNM classification, 46 patients (85%) were stage pT2N0M0 and eight patients (15%) stage pT3N0M0, no significant correlation being found with PSA values. All patients were submitted to pre- and postmicturition retrograde cystourethrography, four weeks after surgery; patients presenting dysuric symptoms also underwent further retrograde cystourethrography and flowmeter examinations. RESULTS: Of the 54 patients, 18 (33%) presented stenosis of the new bladder neck as a complication of radical prostatectomy. Of these 18 patients, 12 (22%) presented acute dysuria symptoms. In 14 cases, one-two cycles of urethral dilation were sufficient to improve flowmeter values. In the four remaining cases showing no improvement following urethral dilations, endoscopic resection was necessary in two and urethrotomy according to Sachse in the other two. CONCLUSIONS: Stenosis of the anastomosis of the new bladder neck is a complication of radical prostatectomy occurring within six months of surgery, no correlation being found with tumour stage, recurrence, or duration of catheter in situ. Of the 18 patients presenting stenosis in the series described, in 78%, cycles of urethral dilation were sufficient to successfully resolve the complication thus avoiding further surgery, which, on the other hand, was necessary in four patients, two submitted to endoscopic resection of the stenosis and the other two to incision according to Sachse.

[Incidence and treatment of postprostatectomy urinary incontinence. Personal experience]. / Incidenza e terapia dell'incontinenza urinaria postprostatectomia. Nostra esperienza.

BACKGROUND: To evaluate frequency of postprostatectomy urinary incontinence in order to establish the last invasive and most efficacious treatment to completely restore urinary incontinence. METHODS: Between 1992 and 2000, twenty-four patients with retropubic postprostatectomy urinary incontinence were studied. The symptoms reported by all patients referred to lack of control of urine with consequent leakage upon activities exerting increased abdominal pressure (sneezing, lifting of heavy weights). These patients were submitted to urodynamic examinations and the degree of incontinence was further evaluated from the number of pads used daily (slight, 0-1; medium 2-3; severe, >3). Mean follow-up was three years, eight months. RESULTS: Of the twenty-four patients, twenty (83%) presented stress incontinence, two (8.5%) urge incontinence, and two (8.5%) a mixed type incontinence. Eighteen patients (36%) reported slight precocious incontinence which disappeared spontaneously within three-six months. Six patients (12%) reported total incontinence which had not improved within twelve months, in four of these patients, continence was achieved by means of perineal rehabilitation whilst in the remaining patients, use of pads (up to three pads/day) was necessary, due to failure of rehabilitation. CONCLUSIONS: Once the need has been established for radical anatomic prostatectomy which offers the possibility of postoperative continence, perineal rehabilitation represents the first choice treatment on account not only of the high percentage of successful results but also due to low invasiveness, whilst the use of the AMS 800 sphincter offers the only solution in those forms of severe incontinence refractory to less invasive forms of treatment.

[Postprostatectomy erectile dysfunction]. / Deficit erettile postprostatectomia.

BACKGROUND: To evaluate the frequency of erectile dysfunction in patients submitted to radical prostatectomy due to prostate carcinoma and to the possibility, with appropriate treatment, of restoring adequate erections for a satisfactory sexual activity. METHODS: Between 1996 and 2000, thirty-eight patients with prostate carcinoma have been submitted to radical prostatectomy. According to TNM classification upon diagnosis, thirty-three patients (87%) were stage pT2N0M0 and five patients (13%) stage pT3N0M0. Mean age of patients was 62.4 years (range 55-76). Mean follow-up was two years and seven months. Diagnosis of erectile dysfunction was made from personal history as well as that of the patient plus partner, and from nocturnal penile tumescence evaluation with Rigiscan. RESULTS: Eighteen patients (47%) presented erectile dysfunction as a complication 16 (89%) of whom showed no nocturnal reaction at the Rigiscan test, while in the remaining two (1%), one-two nocturnal erections of less than five minutes were obtained. Of the eighteen patients, 14 (78%) showed a positive response to treatment with intracavernous drug infusion (papaverine+Phentolamine+Alprostadil), whilst only one patient benefited from treatment with oral Sildenafil. CONCLUSIONS: This study showed the high frequency (47%) of erectile dysfunction in patients submitted to radical prostatectomy as well as the need to perform radical nerve-sparing surgery (particularly in the neoplastic forms not involving the prostate capsule) which is useful not only for preserving erectile function but also on account of better response to oral Sildenafil treatment following the use of this technique.

Cleavage of Bcl-2 in oxidant- and cisplatin-induced apoptosis of human melanoma cells.

Although the anti-apoptotic effect of Bcl-2 is well established, the role of Bcl-2 in tumour response to therapy and drug resistance is still unclear. The post-translational modifications of Bcl-2 are likely involved in the control of the apoptotic pathway. In the present study we have investigated the role of Bcl-2 in cellular response to oxidative stress (hydrogen peroxide) and cisplatin using a clone of human metastatic melanoma, which, in spite of Bcl-2 (over)expression, exhibited a moderate chemosensitivity. With both treatments melanoma cells died through an apoptotic process, associated with detachment of cells from the monolayer. In the floating apoptotic cells generated by either hydrogen peroxide or cisplatin, along with morphological and biochemical features of apoptosis, we detected a significant Bcl-2 cleavage, yielding the Bax-like fragment of 23 kDa. Preincubation of cells with the caspase-3/-7 inhibitor DEVD-CHO completely suppressed Bcl-2 cleavage, thus confirming that such a specific proteolysis requires activation of caspase-3/-7. The oxidant- and cisplatin-induced processing of Bcl-2 documented in the present study may represent a regulatory mechanism to circumvent the survival function of Bcl-2 upon apoptosis triggering and to enhance apoptotic response. Since the Bcl-2 cleavage should be regarded as a pro-apoptotic event, Bcl-2 expression is expected to increase susceptibility to apoptosis. Thus, such a pathway could be exploited to improve the efficacy of cytotoxic therapy of melanomas expressing Bcl-2.

Membrane gamma-glutamyl transpeptidase activity of melanoma cells: effects on cellular H(2)O(2) production, cell surface protein thiol oxidation and NF-kappa B activation status.

The metabolism of glutathione by membrane-bound &ggr;-glutamyl transpeptidase (GGT) has been recently recognized as a basal source of hydrogen peroxide in the extracellular space. Significant levels of GGT activity are expressed by malignant tumours, and in melanoma cell lines they were found to correlate with the malignant behaviour. As hydrogen peroxide and other oxidants can affect signal transduction pathways at several levels, the present study was aimed to verify: (i) the occurrence of GGT-dependent production of hydrogen peroxide in melanoma cells; (ii) the effects of GGT-dependent prooxidant reactions on known redox-sensitive cellular targets, i.e. protein thiols, the nuclear transcription factor NF-kappa B and p53. Two melanoma Me665/2 cell clones, exhibiting traces of (clone 2/21) or high (clone 2/60) GGT activity, were studied. The occurrence of GGT-dependent production of hydrogen peroxide was apparent in 2/60 cells, in which it was accompanied by lower levels of cell surface protein thiols. In 2/60 cells, GGT expression was also associated with higher levels of NF-kappa B activation, as compared to GGT-poor 2/21 cell clone. Indeed, stimulation or inhibition of GGT activity in 2/60 cells resulted in progressive activation or inactivation of NF-kappa B, respectively. An analysis of the p53 gene product indicated lack of protein expression in 2/60 cells, whereas a mutant protein was highly expressed in 2/21 cells. Taken together, these results indicate that the expression of GGT activity can provide melanoma cells with an additional source of hydrogen peroxide, and that such prooxidant reactions are capable to modify protein thiols at the cell surface level. In addition, GGT expression results in an up-regulation of the transcription factor NF-kappa B, which could explain the higher metastatic behaviour reported for GGT-rich melanoma cells as compared to their GGT-poor counterparts.

Site-selective laser spectroscopy of lanthanide-binding sites in calmodulin.

Site-selective laser spectroscopy has been used to resolve the spectral features of lanthanide fluorescence probe ions in calcium-binding proteins. The capabilities and characteristics of this technique are studied using bovine brain calmodulin where the calcium-binding sites are very similar. Two distinct spectral features are identified. These features were followed during a Eu3+ titration and were found to fill successively, showing they correspond to the high- and low-affinity sites. One set of spectral features is assigned to domains I and III, which are the high-affinity domains, while the other set is assigned to domains II and IV. Additional nonspecific binding is observed after the domains are filled. Tb3+ titrations confirmed earlier results that the tyrosine-containing domains fill second and third (R. W. Wallace, E. A. Tallant, M. E. Duckter, and W. Y. Cheung, 1982, J. Biol. Chem. 257(4), 1845-1854). Site-selective laser spectroscopy was also used to identify the presence of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid contamination that could cause interference in titrations.