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Biomedical subjects

G Graziani

Publications and source records attributed to G Graziani.

At least 55 records · Page 3Linked to original sources
PubMed1997

Decline in telomerase activity as a measure of tumor cell killing by antineoplastic agents in vitro.

Telomerase activity is frequently associated with the malignant phenotype, and it can be considered an almost ubiquitous tumor marker. In this study, we evaluated telomerase activity in telomerase-positive human tumor cell lines exposed in vitro to antineoplastic agents. The results show that drug-induced cell killing of tumor cells is associated with a decline in detectable telomerase activity. The decrease of telomerase activity levels paralleled cell growth impairment evaluated by cell count or by measurement of cell ability to convert tetrazolium salt to colored formazan [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide assay]. The observed telomerase activity remaining after treatment with antineoplastic agents is most likely to reflect activity from the remaining viable cells. When tumor cell lines resistant to the chemotherapeutic agents temozolomide or doxorubicin were treated with these compounds, no decline of telomerase activity or cell growth was observed. The results of the present study indicate that resistance of neoplastic cells to chemotherapeutic agents can be monitored by following telomerase activity. Moreover, the test can be performed with a limited number of neoplastic cells, such as those frequently obtained from tumor biopsies. These findings provide a rationale for developing new in vitro chemosensitivity assays, and detection of telomerase activity may be a novel marker of chemotherapy failure.

Antineoplastic Agents↗
PubMed1996

Proteinuria and blood pressure as causal components of progression to end-stage renal failure. Northern Italian Cooperative Study Group.

AIMS: To identify the prognostic factors possibly related to end-stage renal failure development. SUBJECTS AND METHODS: The prognostic factors affecting chronic renal failure progression were analysed in 456 patients who had participated in a formal, multicentre, prospective randomized trial aimed at verifying the role of protein restriction in slowing down or halting the progression of chronic renal failure. The 24-month follow-up foreseen by the protocol was completed by 311 patients and 69 reached an end-point. An inductive analysis on patient survival was made by using the Cox proportional hazard regression model, using a stepwise procedure in order to select only those factors which are significantly associated with survival. For each individual risk factor, a univariate descriptive analysis of survival was performed using the Kaplan-Meier technique. RESULTS: Underlying nephropathy, baseline plasma creatinine, proteinuria, and plasma calcium were all shown to be related to end-stage renal failure onset. Hypertensive patients (mean blood pressure > 107 mmHg) had a worst cumulative renal survival but the degree of proteinuria was even more important as a prognostic factor of renal death than hypertension. The cumulative renal survival of patients whose proteinuria decreased during the trial follow-up was better than those of patients without changes. However, the interaction between baseline lying mean blood pressure and proteinuria was not significant. CONCLUSIONS: Only primary renal disease and proteinuria were related to renal survival, being baseline plasma creatinine confounding factor. By blocking the possible causal role of proteinuria and hypertension, end-stage renal failure could be prevented in a significant percentage of patients.

Adolescent↗
PubMed1996

Development of a novel in vitro chemosensitivity assay: telomerase as a possible marker of tumor cell survival.

Telomerase is a ribonucleoprotein enzyme which is involved in the maintenance of chromosome ends. Telomerase activity (TLMA) is required for cell immortality and can be considered a ubiquitous tumor marker. In this study we describe a new approach for developing in vitro chemosensitivity assays based on the assessment of TLMA in tumors, upon treatment with antineoplastic agents. The results of preliminary studies on in vitro cultured tumor cell lines indicate that TLMA inhibition can be used as a marker of tumor cell killing by anticancer drugs. Therefore the present investigation provides the rational basis for an in vitro chemosensitivity assay based on TLMA evaluation.

Antineoplastic Agents↗
PubMed1996

In vitro inactivation of human O6-alkylguanine DNA alkyltransferase by antitumor triazene compounds.

The cytotoxic and mutagenic properties of antitumor triazene compounds (TZC) have been mainly attributed to their ability to form DNA adducts at the O6 position of guanine. Repair of these lesions is mediated by O6-alkylguanine DNA alkyltransferase (OGAT) in an autoinactivating reaction. Therefore when lesion repair has occurred, cells are depleted of OGAT until synthesis of new enzyme molecules takes place. In this study, we have evaluated the ability of DNA alkylated by different TZC to deplete OGAT activity. Moreover, we have also investigated whether these compounds might inactivate the OGAT enzyme by a direct reaction with the protein. Human OGAT protein was partially purified from insect cells infected with a recombinant baculovirus containing the human OGAT coding sequences. Thereafter human OGAT protein was exposed directly to TZC or to TZC-alkylated DNA. Among the TZC tested, p-(3-methyl-1-triazeno)benzoic acid was the most effective OGAT inactivator by direct interaction with the protein. Moreover DNA substrates treated with methylating TZC, such as temozolomide or p-(3-methyl-1-triazeno)benzoic acid, were more effective in depleting the repair enzyme, compared to DNA pretreated with the chloroethylating TZC mitozolomide. In conclusion, our results show that TZC inactivate in vitro OGAT activity by either direct or indirect mechanisms. Therefore TZC are good candidates for 1) increasing their own cytotoxicity, if used according to appropriate dose and treatment schedules and 2) reversing tumor cell resistance to O6-guanine alkylating agents.

Animals↗
PubMed1996

Drug-induced changes of carcinoembryonic antigen expression in human cancer cells: effect of 5-fluorouracil.

Previous studies showed that 5-fluorouracil (5-FU) is capable of enhancing the membrane reactivity of the human colon carcinoma cell line HT-29 with a monoclonal antibody (COL-1) directed against carcinoembryonic antigen (CEA). In the present study, we show that short-term exposure (i.e., 1 hr) of cancer cells to 5-FU mediates a marked increase of CEA expression, that is concentration-dependent and lasts up to day 5 after treatment. This phenomenon is the result of the drug-mediated enhancement of the CEA expression, but not of the selection of the CEA-positive cells operated by the antimetabolite. This is supported by the finding that the increase of the CEA expression detected by cytofluorimetric analysis is observed not only in the parental HT-29 line, but also in its C22.20 subclone, endowed with a low basal level of CEA and with chemosensitivity to 5-FU lower than that of the parental cell line. Moreover, increase of CEA expression occurs not only in the plasma membrane, but also in the cytosolic cellular compartment, as indicated by the results of Western blot analysis. Northern blot analysis of total RNA extracted from 5-FU-treated HT-29 or C22.20 cells shows an increase in the steady-state levels of CEA and CEA-related transcripts (e.g., biliary glycoprotein). Moreover 5-FU-mediated augmentation of the CEA transcript appears to be attributable mainly to enhanced transcription rather than to increased mRNA stability. It is concluded that induction of enhanced CEA protein expression in cancer cells treated with 5-FU could be of clinical interest for the development of immunochemotherapeutic protocols based on CEA protein as the target molecule.

Carcinoembryonic Antigen↗
PubMed1995

Chimeric molecules between keratinocyte growth factor and basic fibroblast growth factor define domains that confer receptor binding specificities.

Basic fibroblast growth factor (FGF) and keratinocyte growth factor (KGF) are structurally related fibroblast growth factors, yet they exhibit distinct receptor binding specificity. Basic FGF binds with high affinity to FGFR1, FGFR2, and FGFR4, whereas KGF does not interact with these receptors and can only bind an isoform of FGFR2 known as the KGFR. Basic GFG binds KGFR but with lower affinity than KGF. In order to identify domains that confer this specificity, four reciprocal chimeras were generated between the two growth factors and were analyzed for receptor recognition and biological activity. The chimeras are designated BK1 (bFGF1-54:KGF91-194), BK2 (bFGF1-74:KGF111-194), KB1 (KGF31-90:bFGF55-155), and KB2 (KGF31-110:bFGF75-155). The two BK chimera similarly interacted with FGFR1 and FGFR4 but differed from each other with respect to KGFR recognition. BK1 displayed a slightly better affinity for KGFR than BK2 and induced a higher level of DNA synthesis in keratinocytes compared with bFGF and BK2. A neutralizing monoclonal antibody directed against bFGF specifically neutralized the biological activity of the BK chimeras. The reciprocal chimeras, KB1 and KB2, exhibited KGF-like receptor binding and activation properties. However, KB2 displayed higher affinity for KGFR and was significantly more potent mitogen that KB1. Altogether, our results suggest that the amino-terminal part of KGF and bFGF plays an important role in determining their receptor binding specificity. In addition, the results point to the contribution of a segment from the middle part of KGF (residues 91-110) for recognition and activation of the KGFR, as the two chimeras containing these residues (BK1 and KB2) displayed an enhanced interaction with the KGFR.

3T3 Cells↗
PubMed1995

O6-alkylguanine-DNA alkyltransferase attenuates triazene-induced cytotoxicity and tumor cell immunogenicity in murine L1210 leukemia.

Methylating and chloroethylating triazene compounds (TZCs) are effective antitumor agents in murine leukemias and can induce the appearance of novel antigens in leukemic cells (chemical xenogenization). Recently, it has been shown that TZCs might have a role in the treatment of patients affected by acute myelogenous leukemias that express low levels of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (OGAT). In this report, we have evaluated the role of this DNA repair enzyme in the leukemic cell response to the xenogenizing and cytotoxic properties of TZCs. OGAT-deficient murine leukemic L1210 cells were transfected with a recombinant ecotropic retrovirus containing the coding region for the human OGAT protein. Selected clones expressed the human OGAT transcript and had greatly increased OGAT activity. Compared to OGAT-deficient cells, OGAT-expressing cells were considerably more resistant to the xenogenizing properties of 1-(p-chlorophenyl)-3,3- dimethyl-triazene, measured in terms of leukemia graft rejection, and were less susceptible to the cytotoxic activity of the TZCs 8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one and 8-carbamoyl-3-(2-chloroethyl)imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one. These data suggest that methylation of the O6 position of guanine is involved in the appearance of increased tumor immunogenicity after exposure to methylating TZC and that OGAT is able, at least in part, to counteract the cytotoxic effects of methylating and chloroethylating agents.

Animals↗
PubMed1995

Single-tube reverse transcription and heminested polymerase chain reaction of hepatitis C virus RNA to detect viremia in serologically negative hemodialysis patients.

Detection of hepatitis C virus genome is the only method currently available for detecting viremia in infected individuals. We describe a substantial simplification of current methods for viral RNA reverse transcription and polymerase chain reaction amplification, which allows cDNA synthesis and nested amplification in a single tube; this is achieved through compartmentalization of the reagents for the two reactions with a wax barrier. This procedure retains a high sensitivity: analysis of serum samples from hemodialysis patients, a high-risk group for hepatitis C virus infection, confirmed the presence of the virus in 88% of antibody-positive patients and detected viremia in patients without serological or biochemical evidence of hepatitis C virus infection.

Adult↗
PubMed1995

High prevalence of a rare hepatitis C virus in patients treated in the same hemodialysis unit: evidence for nosocomial transmission of HCV.

Hepatitis C virus (HCV) is a major cause of hepatitis among patients treated with maintenance hemodialysis. Blood transfusion appears to be the primary risk factor, but nosocomial transmission of HCV in the dialytic environment has been hypothesized. We addressed this issue by analyzing the individual variation of genomic sequences of HCV in 28 patients on chronic hemodialysis (HD) from the same department and 25 environmentally unrelated patients with HCV-related liver disease. Genome variations of HCV were studied by single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products obtained from the 5'-untranslated region of the viral genome and by sequence analysis. Six different SSCP patterns were identified in HD patients versus 16 in control patients. Among HD patients the three more frequent SSCP patterns accounted for 85% of observations, while in the control group each pattern was found in 4 to 12% of patients. The ability of SSCP to discriminate sequence variation was proven by sequence analysis which confirmed identity/diversity of sequences selected by SSCP. Moreover, sequence analysis permitted a recognition of the most frequent genome observed in HD patients as a type 4 HCV, which is considered to be rare in the Italian population. The relative homogeneity of HCV variants in HD patients treated in the same HD and the high prevalence in this unit of a rare viral variant support the possibility of a nosocomial transmission of HCV in the dialytic environment.

Adult↗
PubMed1995

Hypertension as a factor in chronic renal insufficiency progression in polycystic kidney disease. The Northern Italian Cooperative Study Group.

The aim of this study was to evaluate the role of blood pressure in the progression of chronic renal failure (CRF) in polycystic kidney disease, by analysing the behaviour of 74 affected patients, out of 456 CRF patients with various underlying nephropathies enrolled in a multicentre, formal prospective trial aimed at clarifying the role of protein restriction in retarding CRF progression. Because no difference was found between the patients on a low protein and those on a controlled protein diet, an inductive analysis was made by separating all of the patients into fast progressive or slowly progressive CRF groups. Hypertensive patients were defined as those with a mean resting blood pressure of more than 107 mmHg; of the 62 polycystic patients who completed the study or who reached an end point, 41 patients were hypertensive and 21 normotensive (10 of whom were pharmacologically controlled). The results of the stratified analysis, taking into account the degree of renal function deterioration and the underlying disease, showed a significant relationship between hypertension and CRF progression only in patients with polycystic kidney disease.

Adolescent↗
PubMed1995

Cisplatin increases sensitivity of human leukemic blasts to triazene compounds.

High levels of O6-alkylguanine-DNA-alkyltransferase (OGAT) can, at least in part, account for tumor cell resistance to O6-alkylguanine alkylating agents, including triazene compounds. A pilot clinical study indicates that dacarbazine can induce a marked decrease of leukemic blasts in patients affected by acute myelogenous leukemia (AML) with low pretreatment levels of OGAT activity. In this study we show a synergistic antitumor effect between cisplatin (CDDP) and temozolomide (an in vitro active analog of dacarbazine), following combined in vitro treatment of leukemic blasts. Synergistic effect appears to be CDDP-dose dependent. In vivo treatment of leukemic patients with CDDP was followed by a reduction of OGAT activity in 2 out 3 cases. These data point out that CDDP could be a good candidate for depleting OGAT protein of leukemic cells, thus reversing tumor cell resistance to dacarbazine.

Adult↗
PubMed1995

Triazene compounds induce apoptosis in O6-alkylguanine-DNA alkyltransferase deficient leukemia cell lines.

Previous studies demonstrated that triazene compounds (TZC) possess antitumor, antimetastatic and immunosuppressive activity, and induce novel antigenic properties in neoplastic cells. Moreover, TZC showed marked antitumor activity in patients with acute myelogenous leukemias (AML). In most cases leukemic blasts with low levels of the repair enzyme O6-alkyl-guanine-DNA alkyltransferase (OGAT) were highly susceptible to TZC. Therefore the cytotoxic effects of TZC against human leukemic cells and the influence of OGAT modulation were investigated. Five leukemia cell lines were treated with the in vitro active derivative of dacarbazine: 5-(3-methyl-1-triazeno) imidazole-4-carboxamide (MTIC), or with temozolomide (TZM), which is readily cleaved to form the linear triazene MTIC in aqueous solution. The results showed that treatment with TZC at concentrations ranging between 62.5 and 250 microM significantly inhibited cell growth of U-937 and K-562 leukemia cell lines, both with undetectable OGAT activity. Growth inhibition was accompanied by DNA fragmentation and reduction of cell volume characteristic of cell undergoing apoptosis. In contrast, Daudi, HL-60 and Jurkat leukemia cell lines, characterized by high levels of the repair enzyme, were resistant to concentrations of TZC up to 500 microM. Treatment of resistant lines with O6-benzylguanine (BG, a specific inhibitor of OGAT) rendered HL-60 and Daudi but not Jurkat cells sensitive to cytotoxic effects and apoptosis mediated by MTIC. The results presented suggest that: (1) apoptosis is involved in cytotoxic activity of TZC; (2) OGAT could have a role in preventing programmed cell death induced by TZC; and (3) treatment with BG could potentiate cytotoxic and apoptotic effects of TZC on leukemic cell lines when high level of OGAT activity is the main factor involved in resistance to TZC.

Antineoplastic Agents, Alkylating↗
PubMed1995

Renal effects of dietary supplementation with fish oil in cyclosporine-treated liver transplant recipients.

Nephrotoxicity is the main untoward effect of cyclosporine (CsA) treatment. Experimental and clinical data suggest that dietary supplementation with fish oil may lessen cyclosporine nephrotoxicity, possibly by lowering renal thromboxane (Tx) production. We have studied the renal effects of a daily supplementation for 2 months of 12 g fish oil (18% C20:5 n-3 eicosapentaenoic acid [EPA] and 12% C22:6 n-3 docosahexanoic acid [DHA]) in a placebo-controlled (12 g corn oil), prospective, randomized, double-blind study of stable CsA-treated liver transplant recipients. Thirteen patients ingested corn oil capsules and 13 fish oil. Compliance with dietary regimen was confirmed by fatty acid chromatography that showed increased plasma concentrations of EPA (from 0.4 +/- 0.02% to 4.6 +/- 0.5%, P < .0001) and DHA (from 1.8 +/- 0.2% to 3.9 +/- 0.1%, P < .0001) in the fish oil group and increased plasma concentration of linoleic acid (C18:2 n-6) in the corn oil group (from 25 +/- 2% to 28.4 +/- 2%, P < .001). At the end of the 2 months of the study, in the fish oil group the effective renal plasma flow increased by 22% (P = .012), the glomerular filtration rate increased by 33% (P = .057), the renal blood flow increased by 17% (P = .024), and the calculated total renal vascular resistances decreased by 20% (P = .034). In contrast, none of these parameters changed in the corn oil group. The renal functional reserve determined during L-arginine infusion, plasma renin activity (PRA), and plasma aldosterone (PA) remained unchanged during the study in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Effect of gastric acid secretion on intestinal phosphate and calcium absorption in normal subjects.

BACKGROUND: Phosphate-induced hyperparathyroidism still represents an intriguing problem in dialysis patients. Postprandial hyperphosphataemia is considered to be the main stimulus to parathyroid hyperfunction, and therefore many efforts have focused on the use of phosphate binders to prevent phosphate absorption. METHODS: We investigated whether the pH-mediated gastric ionization of calcium phosphate dietary salts is necessary for its intestinal absorption. In eight normal subjects we measured 24-h urinary calcium phosphate excretion and the postprandial blood calcium phosphate profile after a meal containing 1 g of calcium and 2 g of phosphate salts in a crossover placebo-omeprazole study. On two occasions the subjects received either placebo or omeprazole 60 mg/day 2 days before and during the day test. RESULTS: Serum gastrin levels were measured as an indicator of achlorhydria and were 13.7 +/- 1 pg/ml after placebo and 30.4 +/- 4.7 after omeprazole (P < 0.003). Postprandial plasma phosphate profiles were not significantly different between the two studies (+36 +/- 8% after placebo and +24 +/- 8% after omeprazole, NS), while plasma calcium increased by +6.1 +/- 1% after placebo and decreased by -4.2 +/- 0.7% after omeprazole (P < 0.01). The 24-h urinary phosphate excretion was 1068 +/- 85 mg after placebo and 773 +/- 55 after omeprazole (P < 0.002), while the 24-h urinary calcium excretion was 360 +/- 21 after placebo and 238 +/- 15 after omeprazole (P < 0.0001). A negative relationship was observed between absolute changes in plasma gastrin and those in urinary calcium (P < 0.009) and phosphate (P < 0.05). CONCLUSIONS: The inhibition of gastric acid secretion by omeprazole significantly reduces both urinary phosphate and calcium excretion after an oral load. The behaviour of the postprandial calcium-phosphate plasma profile suggests that gastric acid inhibition is more effective in reducing calcium rather than phosphate dietary salts absorption in normal subjects.

Absorption↗
PubMed1995

In vitro infection of leukemic bone marrow with HTLV-I generates immortalized cell lines expressing T or myeloid cell phenotype.

Leukemic bone marrow cells ( > 90% blasts) of a patient with acute myeloblastic leukemia (AML), non-treated or pretreated in vitro with a mutagenic triazene compound, were infected with HTLV-I by coculture with irradiated virus-donor cells. Immortalized, HTLV-I+, double-positive CD4/CD8 euploid T cell lines, expressing HLA class I/II monomorphic determinants, and inappropriate myeloid and progenitor cell markers (ie CD13, CD14, CD15 and CD33 antigens) were obtained. In one out of 10 triazene-pretreated samples, HTLV-I infection resulted in the appearance of a rapidly growing triploid cell line (ie MTLC1 line) showing: (1) myeloid but not lymphoid phenotype; (2) beta and delta T cell receptor in germline configuration; (3) integrated, complete and incomplete HTLV-I provirus genome (also detected in a number of MTLC1 clones); (4) a high percentage of cells positive for non-specific cross-reacting antigen (a CEA-related molecule present in myeloid cells) under the influence of gamma-interferon; (5) absence of HLA class I/II antigen expression; (6) absence of tax gene transcription. Blast cell proliferation was marginal or absent when leukemic marrow was not subjected to retroviral infection. These results show that exposure of leukemic bone marrow to HTLV-I can be followed by immortalization of T and myeloid cells. Although no data are available to establish whether tax expression played a role in the early phase of the immortalization process of MTLC1 line, tax gene product was not required for maintaining long-term growth of MTLC1 cells.

Antigens, CD↗
PubMed1994

Ocular absorption and distribution of bendazac after topical administration to rabbits with different vehicles.

Ocular and systemic absorption of bendazac was investigated after topical administration to rabbits of 0.5% solutions of bendazac lysine in different polysaccharide vehicles. The results show that the drug is absorbed into the retina-choroid via an extracorneal, or sclero-conjunctival route; the iris and the ciliary body are presumably supplied via both the transcorneal and the extracorneal pathways. The extent of absorption via the extracorneal route is not related to vehicle viscosity but rather to the chemical features of vehicle. The transcorneal penetration appears to be hindered by the binding of the drug to corneal tissues.

Absorption↗