Comparison of topoisomerase-IIalpha gene status between primary breast cancer and corresponding distant metastatic sites

Background. Topoisomerase-II (topo-II) is a key enzyme in DNA replication and a molecular target for anti-cancer drugs called topoisomerase-II inhibitors, such as anthracyclines. Its value as a predictive marker of responsiveness to these cytotoxic drugs is currently being evaluated with promising results. However, even in the metastatic setting, the choice of treatment is based on the biologic characteristics of the primary tumor. Few data are available regarding the expression of biological markers between the primary tumor and the corresponding distant metastases. Methods. Topo-II gene status was evaluated in 29 breast cancer patients in which a primary tumor sample and a corresponding metastatic sample were both available. Fluorescent in situ hybridization (FISH) with the Vysis triple probe (Vysis multi-color topo-II spectrum orange, Her-2 spectrum green and CEP17 spectrum aqua probe) was used, which allowed the concomitant evaluation of HER-2 gene status. Results. As previously reported, topo-II gene aberrations are always associated with HER-2 gene amplification; indeed no topo-II gene aberrations have been observed in the HER-2 negative tumors. Conversely, 38.5% (five patients) of the HER-2 positive primary breast tumors (13 patients) were topo-II amplified, while 61.5% (eight patients) had a normal topo-II gene. No topo-II gene deletion was found in our series. Topo-II gene amplification in the primary tumor was always associated with amplification in the corresponding metastases, and no metastases with topo-II gene amplification were seen without amplification in the primary tumor. Furthermore, the amplification level of topo-II (i.e., ratio topo-II: CEP17) remained unchanged in primary and metastatic sites. Conclusion. Despite the low number of patients, our results seem to indicate that topo-II gene status evaluation in the primary breast tumor accurately reflects its status in the corresponding distant metastases.     

New approach to metabolism of 5′-deoxy-5-fluorouridine in humans with fluorine-19 NMR

Summary The metabolism of 5-deoxy-5-fluorouridine (5dFUrd), an antitumor fluoropyrimidine, has been investigated in human biofluids (blood, plasma, urine) using a new method: fluorine-19 NMR spectrometry. This method allows direct study of the biological sample and simultaneous identification of all the fluorinated metabolites. In the blood of a patient treated with 5dFUrd during a 6-h continuous perfusion, we observed unmetabolized 5dFUrd, 5-fluorouracil, 5,6-dihydrofluorouracil, and another metabolite which has not previously been reported -fluoro--alanine. The two major metabolites in urine are unmetabolized 5dFUrd and -fluoro-alanine.     

Effects of Thalidomide on Parameters Involved in Angiogenesis: An in vitro Study

In an attempt to elucidate the mechanism(s) of action of thalidomide, a reportedly antiangiogenic molecule recently tested in the treatment of relapsing malignant gliomas, we performed an in vitro study on the following parameters: (a) effect of thalidomide on proliferation of endothelial cells; (b) effect of thalidomide on expression of v3 integrin on the surface of endothelial cells; (c) effect of thalidomide on the release by endothelial cells of MMP-2, IL-8 and TNF-. The results show that thalidomide inhibits endotelial cell proliferation induced by bFGF and VEGF, more so if the cells are grown on vitronectin; moreover, treatment with thalidomide reduces the release of MMP-2 and IL-8 by endothelial cells, suggesting a further pathway for the antiangiogenic activity of drug. On the other hand, thalidomide does not modify expression of v3 on endothelial cells.     

Valeur pronostique des mesures de qualité de vie en cancérologie

Résumé: En cancérologie, de nombreuses études ont souligné la valeur pronostique de données issues dévaluations de la qualité de vie (QdV). Lévaluation de la QdV est une mesure des perceptions du patient de limpact de la maladie et des traitements sur son bien-être. Des facteurs pronostiques sont identifiés en relation avec la survie, la réponse au traitement ou la morbidité. La mise en évidence de la valeur pronostique de données de qualité de vie a surtout été effectuée en maladie métastatique; sa valeur se révèle parfois supérieure à celle dindices pronostiques évalués par le clinicien comme lindice de Karnofsky ou létat général OMS. Pour les cancers du sein localisés, les résultats détudes sont hétérogènes. Lexplication la plus plausible du lien entre données de QdV et durée de survie serait que les données de QdV reflètent une perception précoce par le patient de signes subtils de progression de la maladie non relevés par les variables moins nuancées de linterrogatoire médical. Une autre explication porte sur linfluence de processus psychologiques comme loptimisme, lespoir ou un esprit combatif; celle-ci mettrait laccent sur limportance dinterventions psychothérapeutiques favorisant ces dispositions psychologiques pour la survie des patients.Dossier: «Évaluation de la qualité de vie»     

Antitumor activity of 3′, 5′-diesters of 5-fluoro-2′-deoxyuridine against murine leukemia L1210 cells

Summary Antitumor activity of several 3,5-diesters of 5-fluoro-2-deoxyuridine (FUdR) against L1210 leukemia cells following intraperitoneal administration was examined. Esters of FUdR with aromatic acid or aliphatic acid of longer chain length were markedly active. Their activities, with respect to ILS₃₀, were as much as 100 times that of unesterified FUdR. 3,5-ditoluoyl FUdR also had an improved therapeutic effect: its therapeutic ratio was increased to 8.1, as against 2.0 for FUdR. On the other hand, 3,5-diesters of FUdR with aliphatic acid of shorter chain length do not appear to be as active as FUdR. The relationship between the antitumor activity and plasma levels has also been examined. After 3,5-diacetyl FUdR, which is one of the drug group showing low cytotoxicity, the plasma concentration rapidly decreased to unmeasurable level 3 h after dosing. This tendency is similar to that shown in FUdR. On the other hand, with 3,5-dipalmitoyl FUdR and 3,5-dibenzoyl FUdR, each of which has a marked antitumor effect, plasma concentrations decreased slowly and were maintained for as long as 48 h after dosing. The results show that the cytotoxicity of diesters of FUdR is correlated with the duration of a high plasma level of FUdR.     

Immune adjuvants for chemotherapy or radiotherapy in the 9L rat brain tumor model

Summary The therapeutic efficacy and toxicity of three biological response modifiers,Corynebacterium parvum (Cp), Chinese blister beetle extract (CBBE), recombinant human IL-1 (rhIL-1), used alone or in combination with chemotherapy or radiotherapy, were investigated in the intracerebral (ic) rat 9L brain tumor model. Used alone, Cp (2mg/rat, ip plus 70g/rat, ic), CBBE (5l of an ethanol extract, ic), or IL-1 (lg/rat, ic or 1g/rat × 3, q 3 d, ic), had no effect on animal survival compared to the untreated or saline treated controls. When combined with chemotherapy or radiotherapy, the three immunotherapeutic agents did not show any additive effects on survival compared to that observed with systemic BCNU (12mg/kg), local ic bleomycin (0.25 unit), or local radiotherapy (16 Gy). While ic IL-1 did not produce evident toxicity, there was fatal toxicity caused by ic Cp or CBBE treatment in a few animals. The combination of Cp and bleomycin produced severe neurotoxicity, resulting in the early death of animals. This study demonstrates a lack of efficacy of the nonspecific immune adjuvants IL-1, Cp or CBBE, used either alone or combined with cytotoxic chemotherapy or radiotherapy, in this rat brain tumor model.     

Techniques de recherche des polymorphismes génétiques

Résumé: Le séquençage du génome humain est à lorigine de la découverte de millions de variations de séquence dans le génome humain. Ces variations de séquence dADN sont dans leur immense majorité limitées à des polymorphismes de nucléotide unique (Single Nucléotide Polymorphisms ou SNP); cette forme courante de polymorphisme se rencontre environ toutes les 1 000 bases dans le génome humain et 1,8 million de SNP sont actuellement répertoriés. Laccès à ces SNP dans les banques de données publiques a ouvert la possibilité détudier linfluence de ces polymorphismes répertoriés sur la prédisposition à certaines maladies génétiques ainsi que sur la réponse aux médicaments. Lidentification de ces nombreux SNP tient beaucoup aux progrès des techniques danalyse de lADN et nous décrivons, dans cette revue, différentes méthodes pouvant être utilisées pour létude de polymorphismes. Les techniques détude des SNP reposent sur deux principes fondamentaux: dune part la création ou lélimination par le SNP dun site de coupure pour une enzyme de restriction donnée, et dautre part la formation dun mésappariement due à la présence dune séquence variante lors dune hybridation. La technique originelle détude des SNP est le Southern blot couplé à une digestion enzymatique permettant détudier les polymorphismes de taille de fragments de restriction (RFLP). Ce procédé sest largement simplifié par larrivée de la technique de réaction en chaîne par polymerase (PCR). Dautres approches telles que la chromatographie à haute performance en conditions dénaturantes (dHPLC) ou la PCR en temps réel permettent une discrimination des allèles sauvages et variants. En revanche, le séquençage reste la seule technique permettant de déterminer la nature et la position des SNP connus et inconnus. Enfin, lapparition des techniques à haut débit permet de prévoir des changements déchelle considérables dans la recherche des SNP.     

Protection against fludarabine neurotoxicity in leukemic mice by the nucleoside transport inhibitor nitrobenzylthioinosine

Summary Fludarabine phosphate (F-ara-AMP, Fludara) is rapidly converted in the circulation to fludarabine (F-ara-A) and is among the most effective single agents in the treatment of chronic lymphocytic leukemia. Although current treatment protocols are well tolerated, severe neurotoxicity was a consequence of high-dose F-ara-AMP regimens used in early phase I trials against adult acute leukemia. The present study showed that in mice implanted with leukemia L1210, fatal neurotoxicity, which initially manifested as hind-limb paralysis, was a consequence of high-dose-F-ara-AMP treatment. However, the incidence of neurotoxicity was reduced by the coadministration of NBMPR-P, the 5-phosphate of nitrobenzylthioinosine, a potent inhibitor of thees equilibrative nucleoside transport (NT) system. NBTGR-P, the 5-phosphate of nitrobenzylthioguanosine (also a potent NT inhibitor) similarly prevented F-ara-AMP neurotoxicity in this experimental system. Treatment with F-ara-AMP/NBMPR-P combinations was more effective with respect to the fractional yield of cured mice than were the same treatment regimens without NBMPR-P.Abbreviations ara-A 9--d-arabinofuranosyladenine - F-ara-A 9--d-arabinofuranosyl-2-fluoroadenine (fludarabine) - F-ara-AMP fludarabine 5-monophosphate (Fludara) - NBMPR 6-[(4-nitrobenzyl)thio]-9--d-ribofuranosylpurine - NBMPR-P NBMPR 5-monophosphate - NBTGR 2-amino-6[(4-nitrobenzyl)thio]-9--d-ribofuranosylpurine - NBTGR-P NBMPR 5-monophosphate - NBdAdo-P N⁶-(4-nitrobenzyl)-9--d-2-deoxyribofuranosyladenine 5-monophosphate This study was supported by the National Cancer Institute of Canada     

The efficacy of long-term oral chemotherapy with 5′-deoxy-5-fluorouridine and cyclophosphamide for recurrent breast cancer

Background 5-Deoxy-5-fluorouridine (5-DFUR) is a prodrug of 5-fluorouracil (5-FU), which is known to be converted by thymidine phosphorylase (dThdPase). A recent preclinical study revealed that cyclophosphamide (CPA) upregulated dThdPase activity, specifically in tumor cells. The purpose of the present study was to examine the efficacy of long-term administration of 5-DFUR/CPA for patients with recurrent breast cancer.Methods Fifteen breast cancer patients with recurrent tumors entered this study. Ten patients had bone metastasis, five had lung metastasis, and two had liver metastasis. Three patients had multiorgan metastases. All patients had had previous exposure to standard chemotherapy such as CAF (CPA, doxorubicin, and 5-FU) and CMF (CPA, methotrexate, and 5-FU). The patients were orally administered with daily doses of 5-DFUR at 800–1200mg and CPA at 200mg for 2 weeks as induction therapy, followed by 2 weeks rest (one to two cycles). Daily doses of 800mg of 5-DFUR and 100mg of CPA (as maintenance therapy) were continuously administered thereafter. Ten of the 15 patients received the maintenance therapy alone. The treatment was continued for at least 24 months (average, 35.2 months).Results The main findings included a significant decrease in pain in nine patients with bone metastasis, and this effect continued for more than 2 years. As the pain decreased, the patients quality of life (QOL) was improved. Liver metastasis was diminished in two out of two patients. Hematological toxicity of more than grade 3 was recognized in three patients, but only during the induction therapy.Conclusion Oral administration of 5-DFUR/CPA is well tolerated and useful for patients with recurrent breast cancer.     

Characteristics of in vitro antiproliferation activity of human interferon-β

Summary We compared the in vitro antiproliferative activity of highly purified interferon (IFN)- (>10⁷ U protein/mg in antiviral activity) with that of IFNs- and lymphoblastoid, using human cells of malignant and non-malignant origin. IFN- was the least active of three IFNs in suppressing Daudi cell proliferation. Three hematological cells other than Daudi cells cultivated in suspension were insensitive to each of three IFNs. IFN- was more active than IFNs- and lymphoblastoid in suppressing all eight epithelioid cells tested and, particularly with respect to five epithelioid cells sensitive to IFN, IFN- was seven to 49 times as active as IFN-. These results indicate that suppression of cell proliferation by IFN depends not only on the target cell species but also on the IFN species, and emphasize the need for careful selection of the most appropriate IFN species in therapy.We found that the antiproliferative characteristics of the present IFN- preparation were consistent with those reported previously, supporting the idea that IFN- molecules in the present preparation were responsible for suppressing cell proliferation. The antiproliferation activity of our preparation was species-specific but not selective for cells of malignant origin; it was absorbable by IFN-sensitive but not by IFN-insensitive cells; and it was achieved by a cytostatic effect.     

Tolerance of the Normal Canine Brain to Epithermal Neutron Irradiation in the Presence of p-boronophenylalanine

Twelve normal dogs underwent brain irradiation in a mixed-radiation, mainly epithermal neutron field at the Brookhaven Medical Research Reactor following intravenous infusion of 950mg of ¹⁰B-enriched BPA/kg as its fructose complex. The 5 × 10cm irradiation aperture was centered over the left hemisphere. For a subgroup of dogs reported previously, we now present more detailed analyses including dose–volume relationships, longer follow-ups, MRIs, and histopathological observations. Peak doses (delivered to 1cm³ of brain at the depth of maximum thermal neutron flux) ranged from 7.6Gy (photon-equivalent dose: 11.8Gy-Eq) to 11.6Gy (17.5Gy-Eq). The average dose to the brain ranged from 3.0Gy (4.5Gy-Eq) to 8.1Gy (11.9Gy-Eq) and to the left hemisphere, 6.6Gy (10.1Gy-Eq) to 10.0Gy (15.0Gy-Eq). Maximum tolerated threshold doses were 6.7Gy (9.8Gy-Eq) to the whole brain and 8.2Gy (12.3Gy-Eq) to one hemisphere. The threshold peak brain dose was 9.5Gy (14.3Gy-Eq). At doses below threshold, some dogs developed subclinical MRI changes. Above threshold, all dogs developed dose-dependent MRI changes, neurological deficits, and focal brain necrosis.     

Prognostic significance of preoperative MRI scans in glioblastoma multiforme

Tumor necrosis, enhancement, and associated edema in patients with glioblastoma multiforme (GBM) represent biological variables that can be quantitated on preoperative MRI scans. We reviewed 48 highly selected patients, all of whom had supratentorial lesions, had undergone gross total tumor resection, and had received adjuvant treatments (radio- and chemotherapies). None of these patients had had surgery for recurrent tumor resection and none had harbored multifocal tumors. The median age was 50 years. The median Karnofsky performance score was 80. Multivariate analysis using the Cox regression model revealed that the strongest prognostic variable was the amount of tumor necrosis on preoperative scan (P < 0.001), with median survivals of 42, 24, 15, and 12 months for tumor necrosis grades of 0 (7 pts), I (11 pts),11 (9 pts), and III (21 pts), respectively. The intensity of enhancement of the tumor nodule was another prognostic factor (P = 0.003), with median survivals of 35, 18, and 13.5 months for enhancement grades of 0 (2 pts), I (22 pts), and II (24 pts), respectively. The extent of peritumoral edema had a quadratic effect (P = 0.001), with grades I (19 pts), II (22 pts), and III (7 pts) surviving for 24,12, and 20 months respectively. Location and volume of tumors were not statistically significant predictors of survival (P < 0.05). In conclusion, in this highly selected group, GBM patients with little or no necrosis and with less tumor nodule enhancement on preoperative MRI survive longer than patients with greater amounts of necrosis and greater degrees of tumor enhancement. In addition, a moderate degree of peritumoral edema is associated with worse prognosis.     

Conjugation of interferon alpha to a humanized monoclonal antibody (HuBrE-3vl) enhances the selective localization and antitumor effects of interferon in breast cancer xenografts

Human mammary carcinoma xenografts (MCF-7) growing in nude mice were treated with natural interferon (n-IFN-) alone or conjugated to a humanized monoclonal antibody (MoAb) anti-breast mucin (HuBrE-3vl) or to irrelevant human IgG₁. The IFN and the conjugates were administered as 20 intra-lesional (i.l.) injections to 1 of 2 xenografts in each mouse, or i.p. The growth inhibitory effects of HuBrE-3vl/nIFN- were significantly greater than those of nIFN- used as a single agent or conjugated to HuIgG₁. These effects occurred locally in the tumors receiving i.l. injections and systemically, although to a slightly lesser extent, in the noninjected tumors of mice treated i.l. and in the xenografts of mice treated i.p. Biodistribution studies showed that the uptake of ¹²⁵I-HuBrE-3vl/nIFN- by the tumors 24 hours after i.l. or s.c. injection was greater than that of ¹²⁵I-HuIgG₁/nIFN-,¹²⁵ I-nIFN- alone, or by normal tissues, documenting a tumor targeting effect and favorable tumor:normal tissues (T:NT) ratios. The targeting effects and the resulting tumor growth inhibition were favored by the IFN-mediated up-regulation of the HuBrE-3vl reactive antigen, which was more prominent after 3 weeks of treatment with HuBrE-3vl/nIFN-. These results were superior to those we obtained previously with nIFN- conjugated to another MoAb of the same group (Mc5). These studies point out the potential usefulness of HuBrE-3vl/nIFN- for the local and systemic treatment of breast cancer lesions by providing a means of delivering high doses of IFN to the tumors while minimizing the amount of IFN binding to normal tissues.     

Androstenedione and androst-5-ene-3β,17β-diol stimulate DMBA-induced rat mammary tumors — role of aromatase

Summary The effect of the adrenal steroids androst-5-ene-3,17-diol (⁵-diol) and androstenedione (⁴-dione) was studied on the growth of mammary carcinoma induced in the rat by dimethylbenz[a]anthracene (DMBA). The plasma levels of the two steroids were maintained at values within the range of those found in the circulation of post-menopausal women by constant release from osmotic pumps in ovariectomized animals. ⁵-diol and ⁴-dione, at the daily release rate of 500µg, led to plasma levels of 1.26±0.19 and 1.72 ± 0.75 ng/ml, respectively. At these physiologically relevant plasma concentrations, both ⁵-diol and ⁴-dione caused a marked stimulation of tumor growth while having minimal or no effect on uterine weight or on plasma prolactin and LH levels.Concomitant treatment with the aromatase inhibitor aminoglutethimide completely blocked the stimulatory effect of ⁴-dione released from silastic implants on tumor growth, while simultaneous administration of the antiandrogen flutamide had no significant effect. On the other hand, when aminoglutethimide was administered with ⁵-diol, the stimulatory effect of the adrenal steroid on tumor growth was not affected. Such data indicate that, under the present experimental conditions, transformation of ⁴-dione into androgens plays a minor role, the predominant effect of the adrenal steroid being stimulation of tumor growth through conversion into estrogens, while ⁵-diol exerts a direct estrogenic effect independent from aromatase activity. The minimal or absent effect of the same treatment on uterine weight and on plasma prolactin and LH levels indicates the tissue specificity of the effects observed, the mammary tissue being most sensitive to the action of adrenal steroids.F.L. is a MRC Career Investigator.     

Protein Kinase C ζ Isoform is Critical for Proliferation in Human Glioblastoma Cell Lines

Previous studies have confirmed that proliferation in glioblastoma cell lines can be blocked by non-isoform specific protein kinase C (PKC) inhibitors, e.g calphostin C, staurosporine. However, the exact mechanism of PKC involvement is poorly understood. The aim of this study was to explore the role of specific PKC isoforms in the aberrant growth of glioblastoma. Identification of the isoform(s) critical for proliferation in glioblastoma would present a better target for the design of chemotherapeutic strategies. To this end, we screened expression on PKC isoforms in four human glioblastoma cell lines both when proliferating and in a quiescent state using western assays. PKC isoforms , I, II and were found to be expressed in all cell lines. PKC was detected in three out of four cell lines and PKC was detected in one out of four cell lines. Quiescence of growth resulted in down-regulation of PKC. We examined the role of these isoforms by studying the effect of PKC isoform-specific inhibitors bisindolylmaleimide-I and Gö6976 on proliferation in a panel of four human glioblastoma cell lines. Inhibition of PKC and had no effect on proliferation, suggesting that previous studies targeting PKC may not be of therapeutic benefit. More significantly, it was shown that inhibition of PKC blocked proliferation. This suggests that the inhibition of PKC may be an important chemotherapeutic target for arresting growth in glioblastoma.     

«Coût éthique» du cancer et dignité

Résumé: La notion de «coût éthique» du cancer peut surprendre: est-elle recevable et pertinente dans le cadre dune approche humaine et sensible des réalités vécues par la personne malade et ses proches? Au-delà de considérations relevant de la santé publique, des soins, du suivi au cours de la maladie, de laccès aux traitements, on constate que vivre avec le cancer confronte à un cumul de menaces affectant la totalité de lexistence. Le parcours du malade est marqué par lobsédante succession de peurs diffuses, dentraves, daltérations physiques, de pertes, de renoncements, de vulnérabilités que lon peut comprendre comme autant de «coûts» qui simposent à elle. Il semble toutefois nécessaire de privilégier une approche en termes de dignité et de droits ne serait-ce que pour mieux identifier la nature des «coûts» induits par la maladie et concevoir à cet égard les responsabilités qui simposent à tous en termes de sollicitude et de solidarité.     

Reduced cardiotoxicity and increased cytotoxicity in a novel anthracycline analogue, 4′-amino-3′-hydroxy-doxorubicin

Summary The acute and chronic cardiotoxicity and cytotoxicity of the novel doxorubicin (DXR) derivative 4-amino-3-hydroxy-DXR were compared with those of 4-deoxy-DXR and DXR. In the acute cardiotoxicity study, the ECG and hemodynamic changes recorded in anesthetized rats that had been treated i.v. with 10 mg/kg 4-amino-3-hydroxy-DXR or 8.6 mg/kg 4-deoxy-DXR were significantly less severe than those caused by 13 mg/kg DXR. In the chronic cardiotoxicity study, rats received 3 weekly i.v. injections of 3 mg/kg DXR, 3 mg/kg 4-amino-3-hydroxy-DXR, or 2 mg/kg 4-deoxy-DXR during the first 14 days of the study and were observed for an additional 35-day period. DXR induced severe cardiomyopathy that was characterized by ECG changes in vivo (ST-segment widening and T-wave flattening) and by impairment of the contractile responses (F _max,±dF/dt _max) to adrenaline of hearts isolated from treated animals. 4-Deoxy-DXR caused a progressive enlargement of the ST segment in vivo and a significant impairment of the-dF/dt _max value in vitro, which were less severe than those produced by DXR. The least cardiotoxic drug was 4-amino-3-hydroxy-DXR, which induced minor ECG changes without causing significant alterations in the contractile responses of isolated hearts to adrenaline. On the basis of the drug concentration required to inhibit 50% of the colony formation (IC₅₀) of cell lines in vitro, 4-amino-3-hydroxy-DXR was less active than 4-deoxy-DXR but at least twice as active as DXR against human cancer and murine transformed cell lines. These data indicate that 4-amino-3-hydroxy-DXR is significantly less cardiotoxic and more cytotoxic than DXR.     

On the development of an interstitial radiation protocol for a multicenter consortium. Experience with permanent low-dose rate and temporary high-dose rate125I implants in ‘failed’ and ‘newly diagnosed’ glioblastoma patients: Quality assurance methodology and a possible future adjuvant for therapeutic enhancement

Summary Three interstitial implant trial groups (one permanent low-dose rate¹²⁵I and two temporary high-dose rate¹²⁵I implants) in glioblastoma patients (newly diagnosed and failed) were compared to non-randomized similar control groups for efficacy. The results formed the basis for the BTCG 87-01 national implant trial. The pilot trial demonstrated: 1) the effectiveness of a temporary high-dose rate¹²⁵I implant in failed and newly diagnosed patients; 2) the ability of a multicenter consortium to adhere to a standard protocol; 3) a methodology to insure quality assurance; and 4) the possibility of the future adjuvant application of hyperthermia using a single catheter system.     

Acute effects of human recombinant tumor necrosis factor-α on the cerebral vasculature of the rat in both normal brain and in an experimental glioma model

Summary The effects of intravenous (IV) infusion of human recombinant tumor necrosis factor- (rTNF-, Cetus) on normal brain and malignant glioma in rats were examined. Twelve Fischer 344 rats were given either a single injection of 10⁶U rTNF- or injections of 5 × 10⁵U rTNF- for three days. One day post-rTNF- injection (s), rats were injected IV with horseradish peroxidase (HRP) to determine blood-brain barrier (BBB) breakdown and, one hour later, were perfused with an aldehyde fixative and processed for histologic examination. Treatment of normal rats with rTNF- by either dosage or schedule caused no remarkable histopathologic changes in the brain and no alteration in BBB integrity. Human glioma models were produced by intracerebal inoculation of 10⁴ syngeneic RT-2 glioma cells into the right parietal lobe of 30 rats. Animals received single IV injections of 10⁶ U human rTNF- or its excipient (TNF-E) as above on day 3, 7, or 10 post-tumor inoculation or multiple injections of 5 × 10⁵U rTNF- beginning on day 7, 10, or 12 post-tumor inoculation. With a single IV injection of either rTNF- or its excipient, 3-day models showed a similar pattern of HRP extravasation limited to the extracellular space of the tumor inoculation site. In 7-day models treated with a single IV injection of rTNF- or TNF-E, HRP extravasated throughout the tumor, but did not exceed peritumoral margins. In 10-day models treated with a single injection of TNF-E, HRP was found only in the tumor and immediate peritumoral regions while rTNF--treated rats showed more extensive areas of BBB breakdown with HRP evident throughout the entire right hemisphere and extending via the corpus callosum into the contralateral hemisphere. Pericapillary halos were also evident around the small blood vessels within the edematous areas of the corpus callosum. Within tumors, hemorrhagic necrosis and adherence of neutrophils to vessels was observed only in animals treated with rTNF- at 10 days post-tumor inoculation. Multiple IV injections of rTNF- in 7 and 10-day models triggered widespread hemorrhagic necrosis, neutrophil adherence and infiltration in the tumor. There was also extravasation and diffusion of HRP from the site of glioma into the contralateral hemisphere. Twelve-day models treated with multiple rTNF- injections, in addition, showed irregular luminal surfaces and gaps between adjacent endothelial cells of tumor vasculature. These results, which demonstrate that rTNF- can be administered IV in dosages that create widespread necrosis within the glioma but little or no damage to normal brain, suggest that treatment with rTNF- preferentially affects newly-formed vasculature of the tumor and has little effect on the integrity of normal cerebral vessels.     

Metallothionein-like proteins and cell resistance tocis-dichlorodiammineplatinum(II) in L1210 cells

Summary Our studies on the mechanism of resistance of the murine leukemia L1210-PDD line tocis-dichlorodiammineplatinum(II) (cis-DDP) have not shown why it is 10-fold more resistant to the drug than the L1210 line. For this reason we investigated metallothionein-like proteins (MTs) in these cells. Soluble protein extracts from cultures treated for 24 h withcis-DDP, zinc sulphate or saline were anaerobically eluted from columns of chemically reduced Sephadex G-75, and the profiles of zinc, copper and platinum were determined along with those for incorporated radioactive cyst(e)ien and tyrosine. Both salinetreated cell lines contained similar levels of MTs, which were induced by exposure to a minimally toxic level of zinc (100 M). Zinc induction of MTs was nearly 4-fold greater in L1210 than in L1210-PDD cells. The levels of mRNA for metallothionein I (MTI) and (MTII) in uninduced cells were measured by dot-blotting with a cDNA probe. The L1210-PDD cells contained 80% of the MTI and 41% of the MTII compared with L1210 cells, confirming the similar levels in uninduced cells. L1210-PDD cells were 2-fold more sensitive than L1210 cells to cadmium and equally sensitive to zinc. Thus, the resistance of L1210-PDD cells tocis-DDP was not associated with cross-resistance to group II_b metals, whereas their sensitivity to cadmium did reflect the relative inability of the cells to synthesize MTs. The L1210 cells produced MTs when treated with 0.5 and 5.0 M cis-DDP, but the L1210-DDP cells did not when treated with 5.0–40 M cis-DDP. Small amounts of platinum (<21% of the total eluted) were bound to MTs in both cell lines, but platinum provided a minor portion of the MT-bound metals, with zinc and copper contributing the bulk. The basis for the resistance of L1210-PDD cell tocis-DDP is neither an increased level of MTs in the resistant cells nor an enhanced ability to increase the synthesis of MTs after drug exposure.This work was funded from a Peter Crimmins Research Fellowship held by P. G. F.