Transforming growth factor b as a potential tumor progression factor among hyperdiploid glioblastoma cultures: Evidence for the role of platelet-derived growth factor

Among early-passage, near-diploid gliomas in vitro, transforming growth factor type (TGF) has been previously shown to be an autocrine growth inhibitor. In contrast, hyperdiploid ( 57chromosomes/metaphase) glioblastoma multiforme (HD-GM) cultures were autocrinely stimulated by the TGF. The mechanism of this conversion from autocrine inhibitor to mitogen is not understood; previous studies have suggested that platelet-derived growth factor (PDGF) might be modulated by TGF. The similar expression of TGF types 1—3, PDGF-AA, — BB, as well as the PDGF receptor and subunits (a/PDGFR) between biopsies of the HD-GM and near-diploid, TGF-inhibited glioblastomas (GM) by immunohistochemistry did not explain the discrepancy in their regulatory responses. Flowcytometry demonstrated that TGF's mitogenic effect was selective for the aneuploid subpopulations of two of three selected HD-GM cultures,while the diploid cells were inhibited. Among the HD-GM, TGF1 induced the RNA of PDGF-A, c-sis and TGF1. The amount of PDGF-AA secreted following TGF treatment was sufficient to stimulate the proliferation of a HD-GM culture. Antibodies against PDGF-AA, -BB, -AB,PDGFR and/or PDGFR subunits effectively neutralized TGF's induction of DNA synthesis among the HD-GM cell lines, indicating that PDGF served as the principal mediator of TGF's growth stimulatory effect. By comparison, TGF induced only the RNA of PDGF-A and TGF1 among the near-diploid GM; c-sis was not expressed at all. However, the amount of PDGF-A which was secreted in response to TGF1 was insufficient to prevent TGF's arrest of the near-diploid cultures in G₁ phase. Thus, the emergence of hyperdiploidy was associated with qualitative and quantitative differences in TGF's modulation of PDGF-A and c-sis, which provided a mechanism by which the aneuploid glioma cellsmight achieve clonal dominance. We hypothesize that TGF may serve as an autocrine promoter of GM progression by providing a selective advantage to the hyperdiploid subpopulation through the loss of a tumor suppressor gene which mediates TGF's inhibitory effect.     

Selective inhibition of cell proliferation and DNA synthesis by the polysulphated carbohydrate ι-carrageenan

-Carrageenan is a polysulphated carbohydrate that antagonises some heparin-binding growth factors. We assessed the effect of -carrageenan on the proliferation of a panel of cell lines, some of which require heparin-binding growth factors for mitogenesis. The importance of growth factor antagonism for the antiproliferative activity was also determined. Cell proliferation was determined by cell counts and a tetrazolium dye (MTT) assay, and DNA synthesis was determined by thymidine incorporation. The proliferation of the basic fibroblast growth factor (bFGF)-dependent endothelial cell line FBHE was inhibited by daily administration of -carrageenan in a dose-dependent manner [concentration inhibiting cell growth by 50% (IC₅₀ value), approx. 0.5 g/ml]. However, excess bFGF did not reverse the inhibitory effect. DNA synthesis was completely inhibited by concentrations of -carrageenan that nonetheless allowed significant protein synthesis to occur. The proliferation of the androgen-dependent prostate-carcinoma cell line LNCaP was also inhibited by -carrageenan (IC₅₀ value, 5.5 g/ml) and the cells were arrested at the G1/S boundary. -Carrageenan inhibited DNA synthesis in MCF-7 cells stimulated by bFGF and transforming growth factor (TGF) but not in those stimulated by insulin-like growth factor 1 (IGF-1). Blocking IGF-1-mediated DNA synthesis with anti-IGF-1 receptor antibody IR3 enhanced the inhibitory activity of -carrageenan against MCF-7 cells grown in serum. A number of other transformed and non-transformed cell lines were either partially inhibited or not inhibited by -carrageenan. -Carrageenan had low anti-coagulant activity. -Carrageenan is a selective anti-proliferative agent and warrants further investigation for anti-angiogenic therapy (in view of its activity against endothelial cells) and for the treatment of androgen-dependent prostate cancer.     

Ultrastructural Changes of the Vascular Endothelium after Intra-arterial Administration of Tumor Necrosis Factor-alpha (TNFα) in Rat Gliomas

The ensuing ultrastructural changes in tumor vascular endothelial cells following intra-arterial administration of tumor necrosis factor- (TNF) were studied in an experimental rat glioma model. C6 glioma cells were implanted in Wistar rats and then after 14 days, 5×10³U of human natural-type TNF (1.7×10⁵U/m²) was administered through the carotid artery. The animals were sacrificed at 3 or 24h after TNF treatment. A detailed examination with transmission electron microscope revealed swelling of the tumor vascular endothelial cell nuclei and mitochondria with matrix densities at 3h. At 24h, these cells demonstrated the presence of high amplitude mitochondrial swelling or the violent blebbing characteristic of damaged mitochondria; the cytoplasm was swollen enormously and there were dissolution of cytoplasmic organelles and rupture of the plasma membrane. The observed findings were typical of cell necrosis and confirms yet another mechanism by which TNF exerts its anti-tumor effects, that is, necrotizing effects on tumor vascular endothelium. The information appears to be important in the context of clinical application of intra-arterial TNF in the treatment of malignant gliomas.     

Potentiation of the chemotherapeutic action of 5′-deoxy-5-fluorouridine in combination with guanosine and related compounds

Summary The effect of inosine, guanosine, and guanosine 5-monophosphate (GMP) on the antitumor activity of 5-deoxy-5-fluorouridine (5-DFUR) was investigated using P388 leukemia and P815 mastocytoma.The antitumor activity of 5-DFUR was markedly enhanced by coadministration of inosine or guanosine. The increase in lifespan (ILS) of mice treated with 5-DFUR was augmented by the combination with guanosine or inosine in a dose-dependent fashion, and the maximum ILS was about 160% with the combination, while that in the case of 5-DFUR alone was only 48% in the P388 leukemia system. The therapeutic ratio (dose at ILS_max/dose at ILS₃₀) of the combination with guanosine or inosine was 333 and 136, respectively, whereas that of 5-DFUR alone was 3.6. GMP also markedly potentiated the antitumor activity of 5-DFUR in both P388 leukemia and P815 mastocytoma systems, just as it potentiated the activity of 5-fluorouracil in the latter system.The uric acid level in the serum was elevated after IP injection of guanosine or inosine but the value was much lower in the case of guanosine than in inosine.     

Protective effects of fructose-1,6-diphosphate on acute and chronic doxorubicin cardiotoxicity in rats

Summary The effects of fructose-1,6-diphosphate, an intermediate metabolite of glycolysis, on acute and chronic cardiotoxicity of doxorubicin were investigated in rats. In the acute study, urethane-anaesthetized Wistar female rats treated with 10 mg/kg i.v. doxorubicin developed a widening of the ST segment, an impairment of +dP/dt_max, and tachycardia. Pretreatment with 375 and 750 mg/kg i.p. fructose-1,6-diphosphate prevented the ST segment from widening, whereas only 750 mg/kg i.p. significantly attenuated the heart rate increase. Chronic cardiomyopathy was induced over a 6-week period by wekly doses of 3 mg/kg i. v. doxorubicin, being characterized in vivo by the progressive enlargement of the ST segment and the occurrence of histological alterations and in vitro by a marked impairment of the inotropic response elicited by adrenaline in isolated hearts from treated rats. Concurrent treatment with 150 and 300 mg/kg i. p. fructose-1,6-diphosphate thrice a week for 6 weeks did not lessen the chronic heart damage, whereas 600 mg/kg given i. p. significantly reduced the widening of the ST segment and the severity of histological damage in vivo, as well as significantly improving the contractile responses of hearts in vitro. These findings suggest that the administration of fructose-1,6-diphosphate plays a protective role in the acute and chronic cardiotoxicity of doxorubicin in the rat.Supported in part by grants from the National Research Council (CNR), Rome, Italy: (1) target project Medicina Preventiva e Riabilitativa, grant 87.00253.56; (2) target project Oncologia, grant 87.01184.44     

Additive effect of mifepristone and tamoxifen on apoptotic pathways in MCF-7 human breast cancer cells

MCF-7 cells growing in culture were used to study the mechanism of the antiproliferative activity of the antiprogestin mifepristone, as compared with the antiestrogen 4-hydroxytamoxifen or the combination of both. These steroid antagonists induced a significant time- and dose-dependent cell growth inhibition (cytotoxicity). This inhibition of cell survival was associated with a significant increase in DNA fragmentation (apoptosis), downregulation of bcl2, and induction of TGF1 protein. Abrogation of the mifepristone- and/or 4-hydroxytamoxifen-induced cytotoxicity by TGF1 neutralizing antibody confirms the correlation between induction of active TGF1 and subsequent cell death. The effect of a combination of mifepristone and 4-hydroxytamoxifen on cell growth inhibition, on the increase in DNA fragmentation, bcl2 downregulation, and induction of TGF1 protein was additive and significantly different (P < 0.05) from the effect of monotherapy. A translocation of protein kinase C (PKC) activity from the soluble to the particulate and/or nuclear fraction appeared to be also additive in cells treated with a combination of both 4-hydroxytamoxifen and mifepristone. These results suggest that the mechanism of the additive antiproliferative activity of mifepristone and tamoxifen could be explained at least in part by an additive induction of apoptosis in both estrogen and progesterone receptor positive MCF-7 breast cancer cells. A bcl2 downregulation, the PKC transduction pathway, and TGF1 expression seem to be involved in this additive mechanism of action. Our data further suggest that a combination of an antiprogestin with tamoxifen may be more effective than tamoxifen monotherapy in the management of human breast cancer.     

Effect of Dose and Infusion Time on the Delivery of p-boronophenylalanine for Neutron Capture Therapy

Clinical trials of boron neutron capture therapy (BNCT) for glioblastoma multiforme are currently in progress using p-boronophenylalanine (BPA) as the 10B delivery agent. Enhancement of tumor boron uptake and/or the tumor-to-blood (T:B) boron concentration ratio would have the potential of significantly improving the therapeutic gain of BNCT. The effects of total dose, infusion time, and route of administration of BPA on tumor and blood boron concentrations were studied in rats bearing the 9L gliosarcoma. Increasing the total dose of BPA from 250 to 1000mg/kg, administered intravenously over a 2-h infusion period, resulted in an increase in tumor boron concentration from 30 to 70µg 10B/g, with a constant T:B boron concentration ratio of about 3.7:1. Similarly, extension of the infusion time from 2 to 6h, at a constant dose-rate of 125mg BPA/kg/h, resulted in an increase in tumor boron concentration from 30 to 80µg 10B/g, while, again, maintaining a constant T:B ratio of about 3.7:1. In contrast, intracarotid infusion of BPA for 1h at a dose rate of 125mg BPA/kg resulted in an increase in the tumor boron concentration from 26 to 38µg 10B/g with a corresponding increase in the T:B ratio from 3.5:1 to 5.0:1. The effects of these results on the therapeutic gain potentially achievable with BNCT are discussed.     

High-dose chemotherapy with autologous stem cell rescue for children with high risk and recurrent medulloblastoma and supratentorial primitive neuroectodermal tumors

Current treatment for high risk and recurrent medulloblastoma (MB) and supratentorial primitive neuroectodermal tumors (stPNET) has a very poor prognosis in children. High dose chemotherapy (HDCT) and autologous stem cell rescue have improved survival rates. We present 19 patients (thirteen classified in the high risk group and six patients with recurrent disease) that received HDCT and autologous stem cell rescue.In the high risk group [Med Pediatr Oncol 38 (2002) 83], all patients underwent neurosurgical debulking. Standard chemotherapy was prescribed in 10 patients. Radiotherapy was given to 4 patients (all older than 4years old). In the recurrence disease group [Childs Nerv Syst 15 (1999) 498], five patients underwent surgery. Radiotherapy was given to those who were not previously irradiated. The HDCT in twelve patients consisted of busulfan 4mg/kg/day, orally over 4days in 6-hourly divided doses and melphalan at a dose of 140mg/m²/day by intravenous infusion over 5min on day –1. Three patients additionally received thiotepa 250mg/m²/day intravenously over 2days and four patients additionally received topotecan 2mg/m²/day over 5days by intravenous infusion over 30min. The other seven patients received busulfan and thiotepa at the same doses.Patients stem cells were mobilized with granulocyte colony-stimulating factor at a dose of 12g/kg twice daily subcutaneously for four consecutive days. Cryopreserved peripheral blood progenitor cells were re-infused 48h after completion of chemotherapy. With a median follow-up of 34months (range 5–93) eight complete responses and one partial response were observed. Three patients died of treatment-related toxicities (15%). The 2 year event-free survival was 37.67±14% in all patients and 57±15% for the high risk group.Therefore we conclude that HDCT may improve survival rates in patients with high risk/recurrent MB and stPNET despite treatment toxicity.     

L’apprentissage de la relation et de l’examen cliniques en cancérologie

Résumé: Malgré la multiplication des examens complémentaires, lexamen et la relation cliniques restent au cur de la pratique médicale. Ils justifient donc un enseignement structuré et attentif. Cet enseignement fait désormais lobjet dune disposition législative fondée sur le respect des patients. Il tient compte dune approche globale, biopsychosociale. Ses objectifs méritent dêtre précisés en vue dune relation de qualité avec des malades en situation difficile. En pratique on insistera sur la dédramatisation dun diagnostic de cancer, sur la façon dapporter de mauvaises nouvelles (diagnostic initial de cancer ou évolution terminale) et sur la pratique des touchers pelviens. Toute cette formation en cancérologie doit sinscrire en cohérence avec une formation clinique générale.     

Effects of supplemental α-tocopherol and β-carotene on colorectal cancer: results from a controlled trial (Finland)

Background:Some epidemiological investigations suggest that higher intake or biochemical status of vitamin E and -carotene might be associated with reduced risk of colorectal cancer. Methods:We tested the effects of -tocopherol and -carotene supplementation on the incidence of colorectal cancer in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, a double-blind, placebo-controlled trial among 29,133 50–69-year-old male cigarette smokers. Participants were randomly assigned to receive -tocopherol (50mg), -carotene (20mg), both agents, or a placebo daily for 5–8 years. Incident colorectal cancers (n=135) were identified through the nationwide cancer registry, and 99% were histologically confirmed. Intervention effects were evaluated using survival analysis and proportional hazards models. Results:Colorectal cancer incidence was somewhat lower in the -tocopherol arm compared to the no -tocopherol arm, but this finding was not statistically significant (relative risk (RR)=0.78, 95% confidence interval (CI) 0.55–1.09; log-rank test p=0.15). -Carotene had no effect on colorectal cancer incidence (RR=1.05, 95% CI 0.75–1.47; log-rank test p=0.78). There was no interaction between the two substances. Conclusion:Our study found no evidence of a beneficial or harmful effect for -carotene in colorectal cancer in older male smokers, but does provide suggestive evidence that vitamin E supplementation may have had a modest preventive effect. The latter finding is in accord with previous research linking higher vitamin E status to reduced colorectal cancer risk.     

Phase I/II trial of high dose mitoxantrone in metastatic breast cancer: the M.D. Anderson Cancer Center experience

Anthracyclines are among the most active agents in metastatic breast cancer. Mitoxantrone demonstrated a different toxicity profile when compared to doxorubicin. We performed a phase I/II study of singleagent highdose mitoxantrone therapy for advanced breast cancer. Nineteen patients who had a diagnosis of metastatic breast cancer received treatment at the M.D. Anderson Cancer Center between June 1986 and December 1987. The patients received escalating doses of mitoxantrone until a maximum tolerated dose (MTD), defined as grade 3 or 4 nonhematologic toxicity or infection, was obtained. The starting dose of 25 mg/m², given by short intravenous infusion, was escalated by 25% in each fivepatient cohort if each patient in the previous cohort tolerated the initial course and 2 or fewer patients reached the MTD. The median cumulative dose of mitoxantrone was 93 mg/m² (range, 25–205) and the maximum single dose was 39mg/m². Myelosuppression was the dose limiting toxicity. The median duration of granulocyte count 250/l was 5–7 days. Four patients (22%) had infections that required hospitalization, 3 patients (17%) had cardiac toxicity. One patient (6%) achieved a complete response, and 3 (17%) had a partial response, with an overall response rate of 22.3%. No apparent doseresponse relationship was observed in our study. The mitoxantrone dosage recommended for phase II studies is 25mg/m² every 3–4 weeks. We conclude that highdose mitoxantrone therapy for metastatic breast cancer was relatively well tolerated but was not associated with a higher response rate than that of standard dose mitoxantrone.     

Response of multicellular tumor spheroids to liposomes containing TNF-α

Summary This publication describes a new model to investigate the influence of tumor necrosis factor- (TNF-) on a three-dimensional glial cell aggregate under defined, standardized, reproducible conditions using the glioma cell line A 172.The cells are initially grown as normal monolayer culture until they reach a cell density of up to 1×10⁶. Subsequently they are grown as spheroids by the liquid overlay technique. Spheroids grown in this way were divided into ten groups of more than 50 cell aggregates. Three groups were coincubated with free TNF- in increasing dosages (100 ng/ml, 200 ng/ml and 1000 ng/ml); three groups were incubated with empty liposomes (0.2 mg/ml, 0.4 mg/ml and 2 mg/ml); three groups received liposomes which had been loaded with TNF-, and one group, which received no treatment, served as control.The diameter of the spheroids ranged from 80 m to 350 m. There was no significant difference in growth between the 3 groups treated with free TNF-. Comparing spheroids treated with TNF- with those which had been coincubated with empty liposomes, there was a significant difference (p<0.001) in growth, which correlated with the amount of liposomes. Similarly, free TNF- had a significantly (P<0.001) stronger growth-inhibiting effect as compared to liposomes loaded with TNF-. Comparing the groups treated with liposomes only to those treated with liposomes loaded with TNF-, the latter exhibited a more marked (although not significantly) growth-inhibiting effect.The preliminary conclusion is that the major growth-inhibiting effect seems to be mediated by the liposomes. This phenomenon is in agreement with results obtained in monolayer cultures.     

Quality of life in the first year after breast cancer surgery: rehabilitation needs and patterns of recovery

Background. Although mortality rates from breast cancer are declining, many breast cancer survivors will experience physical and psychological sequelae that affect their everyday lives. Few prospective studies have examined the rehabilitation needs of newly diagnosed breast cancer patients, and little is known about the predictors of healthrelated quality of life (QOL) in this population.Methods. Between 1987 and 1990, 227 women with early stage breast cancer participated in a prospective longitudinal study in which detailed information was collected through interviews, standardized measures of QOL and psychological distress, and clinical evaluation. Comparisons of physical and treatmentrelated problems were made according to type of surgical treatment. Multivariate regression analysis was performed to examine the predictors of QOL at one year after surgery.Results. Physical and treatmentrelated problems were reported frequently one month after breast cancer surgery, and occurred with equal frequency in women receiving modified radical mastectomy or breast conservation treatment. There were no significant differences in problems reported at one year by type of surgery; however, frequently reported problems include numbness in the chest wall or axilla, tightness, pulling or stretching in the arm or axilla, less energy or fatigue, difficulty in sleeping, and hot flashes. There was no relationship between the type of surgery and mood or QOL. Poorer QOL one year after surgery was significantly associated with greater mood disturbance and body image discomfort one month after surgery, as well as positive lymph node involvement. Although the majority of patients experienced substantial disruptions in the physical and psychosocial dimensions of QOL postoperatively, most women recovered during the year after surgery, with only a minority (<10%) significantly worsening during that time.Conclusions. At one year after surgery, most women report high levels of functioning and QOL, with no relationship between the type of surgery and QOL. Women who reported lower levels of QOL at one year after diagnosis had greater mood disturbance and poorer body image one month after surgery, as well as lower income and positive axillary nodes.     

Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor

Recent studies from our laboratory have revealed that basic fibroblast growth factor (bFGF) selectively inhibits the proliferation of human MCF-7 breast cancer cells. It has also been shown to enhance cis-platinum-induced apoptosis, decrease levels of the anti-apoptotic gene product bcl-2, and increase levels of the cyclin-dependent protein kinase inhibitor p21/WAF1/Cip1. Transforming growth factor beta-1 (TGF₁), a cell growth regulator has been found to have an inhibitory effect on breast cancer cells. The aim of the present study was to evaluate the possible role of TGF₁ in the antiproliferative effects of bFGF in MCF-7 breast cancer cells. We found that exogenous, as well as endogenous (overexpressed) bFGF increased TGF₁ mRNA expression in the cells and enhanced the secretion of TGF₁ into culture medium. However, exogenous addition of TGF₁ neither led to a decrease in bcl-2 nor induced an increase in the levels of p21/WAF1/Cip1 and neutralizing antibodies to TGF₁, did not reverse bFGF-induced G₁ arrest nor the increase in p21/WAF1/Cip1 level. In contrast, antisense oligonucleotides to TGF₁ abrogated the antiproliferative effects and inhibited the induction of p21/WAF1/Cip1 by bFGF in MCF-7 cells. These data suggest that the anti-proliferative effects of bFGF in human MCF-7 breast cancer cells are mediated by endogenous TGF₁, while exogenous TGF₁ does not mimic all the effects of bFGF on these breast cancer cells. These findings provide an important basis for further investigations into the autocrine and paracrine processes that control the growth of breast cancer cells.     

Effect of exogenous epidermal-like growth factors on mammary gland development and differentiation in the estrogen receptor-alpha knockout (ERKO) mouse

The development of the mouse mammary gland requires the interaction between several different ovarian and pituitary hormones such as estrogen, progesterone and prolactin as well as several locally-derived growth factors in the mammary gland such as epidermal growth factor (EGF), transforming growth factor (TGF), amphiregulin (AR) and heregulin (HRG). The focus of this study was to investigate the degree of mammary growth and differentiation in the adult, virgin mammary gland of wild type (wt) and estrogen receptor knockout (ERKO) females that lack estrogen receptor (ER) after reciprocal transplantation into the cleared mammary fat pad of virgin wt or ERKO mice. In addition, we assessed the local response of ERKO mammary tissue to TGF or HRG1 delivered from slow release-Elvax pellets. Our initial results indicated that when we transplanted virgin wt mammary tissue into ERKO mammary fat pads, mammary morphogenesis failed to occur. However, when transplanted virgin ERKO mammary tissue was transplanted into fat pads of virgin or pregnant wt mice, the development and differentiation of lobuloalveoli was readily observed. In addition, treatment of the virgin ERKO mammary gland with TGF or HRG1 stimulated ducts to undergo localized branching and growth and both growth factors induced secretory differentiation as evidenced by the production of milk proteins, caseins and/or whey acidic protein (WAP). The results from this study imply that in ERKO mammary tissue, ERKO ductal epithelium has the capacity to proliferate and differentiate in response to non-estrogenic, morphogenic stimuli.     

The clinical pharmacology of the adenosine deaminase inhibitor 2′-deoxycoformycin

Summary 2-deoxycoformycin (2-dCF; Pentostatin), a stoichiometric inhibitor of mammalian adenosine deaminase (ado deaminase), exhibits immunosuppressive and antilymphocytic activity in animal test systems. A clinical pharmacology/phase I study of 2-dCF administered as a single agent has been completed (18 patients). Dose levels ranged from 0.1 mg/kgx1 to 0.25 mg/kg/dayx5; ado deaminase and 2-dCF were measured spectrophotometrically. Plasma decay curves were bi-exponential ( and t_1/2 values about 1 and 10 h respectively). Recovery of unchanged 2-dCF from urine (48 h) was 32%–48% of the administered drug. Major toxic manifestations were lymphocytopenia (all patients) and urate nephropathy (1 patient, with subsequent patients in the series receiving allopurinol, 300 mg/day). Three partial responses were seen in seven patients with acute lymphocytic leukaemia receiving 0.25 mg 2-dCF/kg/dayx5.     

The α6β1 and α6β4 integrins in human prostate cancer progression

Summary Prostatic secretions are formed by glands composed of basal and luminal cells and surrounded by a basal lamina. The normal basal cells express several integrins (extracellular matrix receptors) including alpha 2, 3, 4, 5, 6, v, beta 1 and beta 4. These integrin units are polarized at the base of the cells adjacent to the basal lamina. The integrin alpha 6 beta 4 is associated with hemidesmosomal-like structures.The natural history of prostate cancer involves the presence of prostatic intraepithelial neoplasia (PIN) lesions (considered precursor lesions), carcinomain situ and invasive carcinoma. Hemidesmosomal proteins and the 31 and 61 integrins (laminin receptors) are retained in the early PIN lesions. Expression of the integrins 2, 4, 5, v and 4 is lost in carcinoma. The 31 and 61 integrins remain associated with invasive carcinoma, the latter being predominant. Integrin expression in carcinoma is diffuse in the plasma membrane and not restricted to the basal aspects of the cell. The 61 integrin is fully functional as judged by an ability to adhere to laminin and contains the wild type 6A cytoplasmic signaling domain. The 61 integrin is a leading candidate for conferring the invasive phenotype in prostatic carcinoma.Tumor cells with high expression of 6 integrin are more invasive when tested in a SCID mouse model system. Following intraperitoneal injection, the human tumor cells invade the mouse diaphragm and move through the muscle on the surface of the laminin coated muscle cells. Our current working hypothesis is that the production of 61 and laminin in human tumor cells contributes to the invasive phenotype. Invasion could occur on the surfaces of laminin coated structures such as the nerves, blood vessels or muscle and account for the known patterns of human prostate tumor progression. Blockage of the expression or function of 61 or laminin or preventing the loss of 4 would be essential steps in confining the carcinoma to the prostate gland where conventional treatment has already proven effective.     

Expression of TGFα in Meningiomas

The objective of this study was to examine the expression of transforming growth factor (TGF), a mitogen for many cell types, and its receptor in basic subtypes of meningiomas as well as in meningiomas of varying grade. Formalin-fixed tissues from 26 meningiomas including 15 benign (5 meningothelial, 5 transitional, and 5 fibrous variants), 6 atypical, and 5 malignant examples were immunohistochemically examined for both TGF protein and EGF/TGF receptor protein. In addition, in situ hybridization (ISH) was used to detect TGF mRNA expression. Immunostaining for TGF was strongest in fibrous and atypical meningiomas, followed closely by transitional and malignant tumors. Only weak reactivity was observed in the meningothelial variant. In all but 4 tumors (2 fibrous, 2 atypical), ISH showed TGF mRNA to be present, the signal being stronger in malignant than in conventional or atypical tumors. Lastly, immunostaining for EGF/TGF receptor was positive in all tumors studied. Strong TGF protein expression in meningiomas is commonly associated with fibrous morphology. Although the frequent detection of both TGF protein and its mRNA, as well as of EGF/TGF receptor within tumors of all type and grades, suggests that TGF serves to promote tumor growth, its possible role in tumorigenesis or malignant progression is uncertain.In summary, demonstration of these substances is of no utility in the classification or grading of this common tumor because the differences in their expression among the various meningioma subtypes were not statistically significant.     

Role of Ceramide During Cisplatin-induced Apoptosis in C6 Glioma Cells

Cisplatin is commonly used for the treatment of malignant brain tumors. However, the mechanisms of cell death by cisplatin are not fully understood. Therefore, the present study was designed to elucidate the apoptotic signaling pathway(s) activated by cisplatin in a C6 rat glioma cell line. C6 cells were treated with various concentrations of cisplatin (0.2–10g/ml) for 24–72h. At 10g/ml cisplatin, over 90% of the cells became dead at 72h. Apoptotic death was confirmed by condensation and fragmentation of nuclei, and DNA laddering. Even in cells treated with 1.5g/ml cisplatin, typical apoptotic cells were observed at 72h. The intracellular level of ceramide, measured Escherichia coli diacylglycerol kinase markedly increased during 24–72h after the addition of 10g/ml cisplatin. The activity of caspase-3(-like) proteases increased and reached a peak at 48h. Inhibitors of caspases reduced the number of apoptotic cells. Pretreatment of C6 cells with glutathione or N-acetyl-cysteine, which are known to block the activation of neutral magnesium-dependent sphingomyelinase, inhibited ceramide formation, leading to suppression of both activation of caspase-3(-like) proteases and apoptosis by cisplatin. In contrast, pretreatment of the cells with N-oleoylethanolamine (OE), a ceramidase inhibitor, potentiated apoptosis induced by cisplatin. Furthermore, OE enhanced sensitivity of the cisplatin-resistant cells to cisplatin. These results suggest that ceramide is closely implicated in apoptosis of glioma cells by cisplatin through activation of caspase-3(-like) proteases.     

Phase I/II tolerability/pharmacokinetic study with one-hour intravenous infusion of doxifluiridine (5′-dFUrd) 3 g/m2 VS 5 g/m2 QD x 5 per month

Summary Eighteen patients with advanced solid cancer were treated with daily 5-dFUrd infusions given over 1 h on days 1–5 of a 4-week cycle. Nine patients received 3 g/m² 5-dFUrd daily and another nine patients 5 g/m². One patient on 5 g/m² 5-dFUrd was not fully evaluable for tolerability due to early death (progressive disease) 4 weeks after the first cycle. A total of 48 cycles was given. The gastrointestinal and hematological toxicity was generally mild (grade 1–2). Central neurotoxicity (ataxia, unsteadiness, diplopia, dysarthria, sometimes confusion) was observed in 7 of 8 patients on 5 g/m² 5-dFUrd leading to premature discontinuation of treatment in 3 patients (after 2 cycles). Only 3 of the 9 patients in the 3 g/m² group had slight signs of cerebellopathy. Typically, the reversible neurological side effects started at the end of the 2nd week of a cycle. The serum elimination kinetics of 5-dEUrd and its metabolites 5-FU and 5-dFUH₂ have been investigated in the serum and showed very low intra- and interindividual variations. Peak concentrations of the 5-dFUrd at the end of the infusion approximated 500 mol/l and 1000 mol/l for the 3 g/m² and 5 g/m² group, respectively. The peak of the serum 5-FU was reached at the same time, the ratio 5-FU/5-dFUrd being around 10%. The elimination half-life time for 5-FU was protracted by a factor of 2–3 compared with the direct injection of 5-FU.Monthly infusion of 5-dFUrd 5 mg/m² per day on days 1–5 lead to an unacceptable frequency and degree of neurological toxicity. Similar infusions of 5-dFUrd 3 g/m² per day on days 1–5 were well tolerated.