Derivation and initial characterization of a mouse mammary tumor cell line carrying the polyomavirus middle T antigen: utility in the development of novel cancer therapeutics.

Here we describe the derivation of novel cell lines from spontaneous mammary tumors that arose in mouse mammary tumor virus-polyomavirus (MMTV-PyV) Middle T (MidT) transgenic mice. Clonal cell lines from four mixed cell populations were tested for adenovirus transducibility and sensitivity to p53 tumor suppressor gene therapy mediated by SCH58500, a replication-deficient adenovirus that expresses human p53. The MidT2-1 cell line was selected for further characterization in vitro and in vivo. This cell line carried the PyV MidT antigen, had wild-type p53 DNA, and was sensitive to suppression of proliferation by MMAC/PTEN tumor suppressor gene therapy. MidT2-1 cells gave rise to highly aggressive tumors in syngeneic FVB mice in both the mammary fat pad and the peritoneal cavity. The histopathology of MidT2-1 tumors closely resembled the histopathology of the primary transgenic tumors. Tumor growth in vivo was inhibited by p53 gene therapy or by MMAC gene therapy. In addition, combination therapy with a number of anticancer agents had synergistic or additive efficacy in vitro. In particular, MMAC gene therapy synergized with SCH58500 or paclitaxel. In the i.p. MidT2-1 tumor model p53 gene therapy enhanced the survival benefits of paclitaxel/cisplatin chemotherapy. Combination therapy has become a mainstay in cancer treatment. In this report, we use a novel transgenic mouse tumor cell line to suggest new combinations that might be explored in clinical cancer care. These include gene therapy using the tumor suppressors MMAC and p53, chemotherapy using farnesyl transferase inhibitors, the microtubule stabilizing taxanes, and the DNA synthesis disruptors gemcitabine and cisplatin. The precise biological mechanisms by which these therapies induce their antitumor effects are not fully elucidated. However, the work presented here suggests that many of these therapeutic approaches have synergistic antitumor activity when used in combination.

Evaluation of endostatin antiangiogenesis gene therapy in vitro and in vivo.

Progressive growth and metastasis of solid tumors require angiogenesis, or the formation of new blood vessels. Endostatin is a 20-kDa carboxy-terminal fragment of collagen XVIII that has been shown to inhibit endothelial cell proliferation and tumor angiogenesis. Replication-deficient recombinant adenovirus (rAd) vectors were constructed, which encoded secreted forms of human and mouse endostatin (HECB and MECB, respectively), and, as a control, human alkaline phosphatase (APCB). Accumulation of endostatin was demonstrated in supernatants of cultured cells infected with the endostatin rAds. These supernatants disrupted tubule formation, inhibited migration and proliferation, and induced apoptosis in human dermal vascular endothelial cells or human vascular endothelial cells. Endostatin-containing supernatants had no effect on the proliferation of MidT2-1 mouse mammary tumor cells in vitro. A pharmacokinetic study of MECB in immunocompetent FVB mice demonstrated a 10-fold increase of serum endostatin concentrations 3 days after intravenous administration of 1x10(10) particles of this rAd (215-257 ng/mL compared to 12-38 ng/mL in control rAd-treated mice). Intravenous administration of MECB reduced b-FGF stimulated angiogenesis into Matrigel plugs by 38%. Intratumoral MECB inhibited growth of MidT2-1 syngeneic mammary tumors in FVB mice, but had minimal impact on the growth of MDA-MB-231 human breast tumors in SCID mice. Intravenous therapy with MECB also initially inhibited growth of MidT2-1 tumors, but this activity was subsequently blocked by induced anti-rAd antibodies. In summary, endostatin gene therapy effectively suppressed angiogenic processes in vitro and in vivo in several model systems.

The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo.

PURPOSE: SCH66336 is an orally active, farnesyl protein transferase inhibitor. SCH66336 inhibits ras farnesylation in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. The taxanes, paclitaxel (Taxol) and docetaxel (Taxotere) block cell mitosis by enhancing polymerization of tubulin monomers into stabilized microtubule bundles, resulting in apoptosis. We hypothesized that anticancer combination therapy with SCH66336 and taxanes would be more efficacious than single drug therapy. METHODS: We tested the efficacy of SCH66336 and taxanes when used in combination against tumor cell proliferation in vitro, against NCI-H460 human lung tumor xenografts in nude mice, and against mammary tumors in wap-ras transgenic mice. RESULTS: SCH66336 synergized with paclitaxel in 10 out of 11 tumor cells lines originating from breast, colon, lung, ovary, prostate, and pancreas. SCH66336 also synergized with docetaxel in four out of five cell lines tested. In the NCI-H460 lung cancer xenograft model, oral SCH66336 (20 mg/kg twice daily for 14 days) and intraperitoneal paclitaxel (5 mg/kg once daily for 4 days) caused a tumor growth inhibition of 56% by day 7 and 65% by day 14 compared to paclitaxel alone. Male transgenic mice of the wap-ras/F substrain [FVB/N-TgN(WapHRAS)69LlnYSJL] spontaneously develop mammary tumors at 6 9 weeks of age which have been previously shown to be resistant to paclitaxel. Paclitaxel resistance was confirmed in the present study, while SCH66336 inhibited growth of these tumors. Most importantly, SCH66336 was able to sensitize wap-ras/F mammary tumors to paclitaxel chemotherapy. CONCLUSION: Clinical investigation of combination therapy using SCH66336 and taxanes in cancer patients is warranted. Further, SCH66336 may be useful for sensitizing paclitaxel-resistant tumors to taxane treatment.

[HCV infection and risk of transfusion in the area of Tivoli and its surroundings]. / Infezione da HCV e rischi trasfusionali nel territorio di Tivoli e dintorni.

The positivity of the anti-HCV antibody has been studied by means of an immuno-enzymatic solid phase method, on 1.605 blood samples. They were drawn from 5 groups of people, during the period from February 1 to October 31, 1992: a) all blood donors who made the donation at the Transfusion Service of Tivoli Hospital during that period; b) all intravenous drug users who came to Tivoli Hospital for control; c) all patients in the Contagious Disease Section with suspected liver disease, always during the same period; d) all patients with suspected liver disease from other Sections of our Hospital; e) all out-patients who came to our Service during the same period to have their hepatitis markers studied. The highest prevalence of HCV Ab positivity was in the drug users, with a prevalence of 80.9%; far from this value, the next two groups were the patients from the Contagious Disease Section (positivity: 23.4%), and from the other hospital Sections (positivity: 20.1%). In the out-patient group only 9.7% were positive and among blood donors only 0.35%. In all 5 groups the HCV-positive subjects were in many cases positive for B hepatitis too; and very often they presented high levels of ALT. These results confirm that in some the percentage of positive-subjects for C hepatitis or for B & hepatitis; very high; therefore the authors underline the great importance to exclude all members of these groups from the donation of blood, its components, and organs too, even if the tests are negative.

Biochemical characterization of the gamma-secretase activity that produces beta-amyloid peptides.

Recent studies of gamma-secretase have pointed out that it may be comprised of a multisubunit complex with presenilin 1 and presenilin 2 as central components. Elucidation of the biochemical mechanism of this enzymatic activity will provide important information for developing gamma-secretase inhibitors in Alzheimer's disease therapy. Here we describe the biochemical characterization of gamma-secretase activities using a sensitive, membrane-based assay system. Membranes were isolated from 293 cells expressing C99, the substrate of gamma-secretase. Upon incubation at 37 degrees C, C99 is cleaved by the endogenous gamma-secretase, and Abeta peptides are liberated. Abeta40 and Abeta42 gamma-secretase activities are very similar in terms of their kinetic profiles and pH dependence, supporting the notion that a single enzyme is involved in both Abeta40 and Abeta42 production. Pepstatin A inhibited Abeta40 and Abeta42 gamma-secretase activities with similar potency. Peptide difluoroketone and peptide aldehyde inhibitors inhibited Abeta40 production in a dose-dependent fashion, enhanced Abeta42 production at low concentrations, and inhibited Abeta42 production at high concentrations. Although the selective increase of Abeta42 by low concentrations of peptide difluoroketone and peptide aldehyde inhibitors has been reported in intact cells, the finding that this phenomenon occurs in a membrane-based assay system suggests that these compounds increase Abeta42 by a direct effect on gamma-secretase. The ability of these compounds to increase Abeta42 production may reflect allosteric modulation of the gamma-secretase complex by a mechanism related to that responsible for the increase of Abeta42 production by mutations in presenilins.

[Effects of betamethasone administration on maternal-fetal Doppler velocimetry]. / Effetti della somministrazione del betametasone sulla velocimetria Doppler materno fetale.

BACKGROUND: The effect of antenatal betamethasone on fetal parameters includes a transient reduction of fetal heart rate variation and of fetal body movements. An effect on maternal-fetal blood flow has also been shown, with non-univocal results. AIMS: To evaluate the effect of antenatal betamethasone in third trimester singleton high-risk pregnancies by Doppler technology. SUBJECTS AND METHODS: Thirty-six pregnant women who received a full course of betamethasone (12 mg i.m. two times, 24 hrs apart) were studied. The Doppler examination included the assessment of the pulsatility index (PI) of the umbilical artery (UA PI), the middle cerebral artery (MCA PI) and of resistance index of uterine arteries (Ut RI) before treatment, and 48 and 96 hrs after second dose of betamethasone. RESULTS: No significant variation was noted in UA PI through betamethasone therapy. MCA PI decreased significantly 48 hours from the last injection of betamethasone (p = 0.004), and returned to basal values at 96 hours. No difference was found for the other Doppler parameters examined. When serial Doppler studies were analyzed according to the gestational age, in the group < 32 wks' gestation, MCA PI decreased significantly after 48 hours (p < 0.006) and returned to pre-treatment values after 96 hours from the last betamethasone dose. Conversely, no difference was found in Doppler serial measurements in any of the analyzed districts in the subgroup > or = 32 wks' gestation. CONCLUSION: Betamethasone treatment is associated with an increase in end diastolic flow of the middle cerebral artery, as reflected by a significant, although transient, reduction of MCA PI, especially at gestational ages < 32 weeks.

Purification and preliminary characterization of a serine hydrolase involved in the microbial degradation of polychlorinated biphenyls.

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA) hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD gene of Burkholderia cepacia LB400, was hyperexpressed and purified to apparent homogeneity. SDS-polyacrylamide gel electrophoresis confirmed that BphD has a subunit molecular mass of 32 kDa, while gel filtration demonstrated that it is a homotetramer of molecular weight 122,000. The enzyme hydrolyzed 6-phenyl-HODA with a kcat of 5.0 (+/- 0.07) s-1 and a kcat/Km of 2.0 (+/- 0.08) x 10(7) M-1 s-1 (100 mM phosphate, pH 7.5, 25 degreesC). The specificity of BphD for other 2-hydroxy-6-oxohexa-2,4-dienoates (HODAs) decreased markedly with the size of the C6 substituent; 6-methyl-HODA, the meta cleavage product of 3-methylcatechol, was hydrolyzed approximately 2300 times less specifically than 6-phenyl-HODA. By comparison, the homologous hydrolase from the toluene degradation pathway, TodF, showed highest specificity for 6-methyl- and 6-ethyl-HODA (kcat/Km of 2.0 (+/- 0.05) x 10(6) M-1 s-1 and 9.0 (+/- 0.5) x 10(6) M-1 s-1, respectively). TodF showed no detectable activity toward 6-phenyl-HODA and 6-tert-butyl-HODA. Neither BphD nor TodF hydrolyzed 5-methyl-HODA efficiently. The kcat of BphD determined by monitoring product formation was about half that determined by monitoring substrate disappearance, suggesting that some uncoupling of substrate utilization and product formation occurs during the enzyme catalyzed reaction. Crystals of BphD were obtained using ammonium sulfate combined with polyethylene glycol 400 as the precipitant. Diffraction was observed to a resolution of at least 1.9 A, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.

[Pulsatility index of the umbilical artery and fetal movements/hour in the prediction of neonatal acidemia in high risk pregnancy]. / Pulsatility index dell'arteria ombelicale e movimenti fetali/ora nella predizione dell'acidemia neonatale in gravidanze ad alto rischio.

OBJECTIVE: The aim of this study was to correlate antepartum computerized cardiotocography (cCTG) and Doppler velocimetry parameters to umbilical blood gas analysis (UBGA) value and Apgar score 1-5 min as neonatal outcome endpoints. METHODS: Forty-eight third trimester single high risk pregnancies were considered for this study, with a cCTG performed within 24 hrs from delivery, a complete Doppler velocimetry study performed within 48 hrs from delivery and an UBGA evaluation at birth and before the first breath. RESULTS: When we analyzed the backward stepwise regression of each Doppler velocimetry and cCTG parameters versus UBGA parameters as dependent variables, we found that the linear combination of fetal heart rate (FHR) (p < 0.001), high variation episodes in min (HV) (p < 0.01) and low variation episodes in msec (LV) (p < 0.03) was able to predict pO2 values. When performing a logistic regression of data for every single parameter of FHR tracing and Doppler velocimetry against pH < 7.2 and Becf < -4 as endpoints, we found that only umbilical artery pulsatility index (UA PI) was able to predict umbilical artery pH (O.R.: 8.1 [1.07-61.8]) and only fetal movements (FM) from FHR tracing values was able to predict UA pH (O.R.: 0.94 [0.89-0.99]). Further analysis considers a cut-off for the prediction of UA pH < 7.2 at birth a value at > or = 1.35 for UA PI and at < or = 12 for FM/h. The combination of UA PI > 1.35 and FM/h < 12 did not improve the ability to predict acidemia at birth. CONCLUSION: The validity of our data from the clinical point of view suggests that in third trimester high risk pregnancies, an UA PI > or = 1.35 and/or FM/h < or = 12 (in a FHR tracing > or = 40 min) may represent a risk of 70% acidemia in neonates.

[Computerized analysis of antepartum fetal heart rate and maternal glycemic levels in pregnancy complicated with insulin-dependent diabetes]. / Analisi computerizzata della frequenza cardiaca fetale (cCTG) antepartum e valori glicemici materni in gravidanze affette da Diabete insulino dipendente.

We aimed to evaluate the effect of maternal glycemia on the parameters of CTG as assessed by a computerized fetal heart rate interpretation system (cCTG). We studied prospectively 22 single insuline dependent diabetic pregnancies (34-39 wks' gestation). A negative correlation was found between high variability epochs (HV) in msec, and maternal glycemia (r = -0.58, p < 0.01). HV epochs are known to be related with hypoxic episodes in the fetus. We suggest that the evaluation of maternal glycemia at the time of cCTG may increase the diagnostic accuracy of automated fetal heart rate interpretation.

[Fetal lung maturity and fetal flowmetry changes]. / Maturità polmonare fetale ed alterazioni flussimetriche fetali.

OBJECTIVE: To verify whether in high risk pregnancies, fetuses with altered Doppler flow velocimetry waveforms (FVW) are associated with both biochemical and/or biophysical changes in the amniotic fluid (AF) lung surfactant. MATERIALS AND METHODS: Hundred-eight high risk pregnancies were considered on the basis of a complete Doppler velocimetry study performed within 24 hrs before amniocentesis and delivery within 1 week from amniocentesis. From this group, 22 pregnancies were recruited as altered FVW cases on the basis of simultaneous alteration of Umbilical Artery Pulsatility Index (UA PI), and UA PI/MCA PI (Middle Cerebral Artery Pulsatility Index) ratio (UA/MCA); in all altered FVW group cases the Mean Uterine Artery Resistance Index (Ut RI) was also altered. Amniotic fluid samples obtained by ultrasound guided amniocentesis were analysed for FLM within 24 hours from collection. Pulmonary maturity was determined by planimetric lecithin/sphingomyelin ratio (L/S), phosphatidylglycerol (PG) and the lamellar bodies count (LBs). RESULTS: When FLM parameters were regressed versus FVW in the whole study group, we found a significant negative correlation between UA/MCA and L/S (r = -0.19, p < 0.05). No significant correlations were found between FVW indices and LBs. When we compared FLM parameters for gestational age subgroups (23-32 wks, > 33 wks), L/S values were significantly higher in patients with normal FVW indices in comparison to pathologic FVW pregnancies for gestational ages > 33 wks. This was reflected by a negativè correlation between FVW and FLM (UA/MCA vs. L/S r = -0.54, p < 0.02; UA/MCA vs. PG, r = -0.60, p < 0.01). COMMENT: Our data suggest that fetuses with an UA/MCA ratio > 95 degrees are not protected from neonatal RDS, as usually felt, but show a delay in FLM, particularly in pregnancies > 33 wks.

[Fetal lung maturity in biamniotic twin pregnancy]. / Maturità polmonare fetale nelle gravidanze gemellari biamniotiche.

OBJECTIVE: To assess the need to perform amniocentesis for the evaluation of fetal lung maturity (FLM) in both amniotic sacs of twin pregnancies. STUDY DESIGN: A retrospective study. We considered 33 twin biamniotic pregnancies, delivering within 1 week from amniocentesis. FLM testing was performed by planimetric lecithin/sphingomyelin ratio (L/S), and lamellar bodies count (LB) according to our methodology. RESULTS: A strong correlation was found between L/S (p < 0.001, r = 0.59) and LB (p < 0.001, r = 0.69) values between smaller and larger twins. No significant regression was found between differences of L/S and LB among both amniotic sacs against gestational age. When stratifying FLM values < 34 and > 34 wks' gestation, again no difference was found in mean difference of L/S and LB before and after 34 wks. CONCLUSIONS: FLM should not be assessed in both amniotic sacs at any gestational age independent of weight and/or sex of the conceptus.