Antiproliferative effects of natural interferon beta alone and in combination with natural interferon gamma on human breast carcinomas in nude mice

Summary Nude mice bearing bilateral xenografts of human breast carcinoma cells (MCF-7 and BT20) were treated with 2 or 4 5-day cycles of intralesional (i.l.) injections of human natural interferon beta (nIFN-) alone or in combination with human natural interferon gamma (nIFN-). The injections were administered to only 1 of the 2 tumors in each animal, thus making it possible to assess at the same time local therapeutic effects in the injected tumors and systemic effects in the contralateral ones. When n-IFN- was used as a single agent only mild local antitumor effects and virtually no systemic effects were observed. In contrast, the combined administration of nIFN-/nIFN- produced marked antiproliferative effects, presumably as a result of the synergistic action of type I and type II IFNs. These effects ranged from complete regression documented histologically in 2 MCF-7 tumors to varying degrees of growth inhibition with persistence of residual microscopic or grossly detectable tumor. Local effects were more pronounced than systemic effects. The therapeutic efficacy of nIFN- proved to be greater than that of recombinant interferon beta (rIFN-). In MCF-7 tumors nIFN- appeared to be less effective than nIFN-, whereas the opposite was true for BT 20 tumors.     

Inhibition of cell proliferation and glutathione S-transferase by ascorbyl esters and interferon in mouse glioma

Summary Mouse glioma-26 (G-26) cell line established in this laboratory was used in the study. Thein vitro effect of ascorbyl esters, viz., ascorbyl-palmitate (As-P), -stearate (As-S) and mouse interferon-/ (MulFN-/) on the glioma cell viability, proliferation and glutathione S-transferase (GST) activity was investigated. Cell viability and proliferation were examined by colorimetric MTT assay and [³H]-thymidine incorporation, respectively. Incubation (24 h) of G-26 cells with As-S, As-P or MulFN-/, resulted in a dose dependent decrease in cell viability (IC₅₀=125M As-S; 175M As-P and 3.6×10⁴ U/ml MulFN-/) and proliferation (IC₅₀=157M As-S; 185M As-P and 3.6×10⁴ U/ml MulFN-/). A combined exposure to 175 M As-S and 800 U/ml of MulFN-/ resulted in a greater than an additive effect on cell viability and proliferation. The inhibition of cell proliferation/viability by interferon was species specific and was observed only with homologous MulFN-/, but not with human interferon- lymphoblastoid or human interferon-. Ascorbyl esters inhibited cytosolic GST activity (1–50=15.0 M As-S and 28.5 M As-P) towards 1-chloro-2,4-dinitrobenzene in a dose dependent manner. The apparent Ki values for affinity purified GST, deduced from Dixon plots were 0.95 M and 2.0 M for As-S and As-P, respectively. Significant inhibition of GST was also observed in the cytosol isolated from G-26 cells exposed to 300 M As-S or 800 U/ml MulFN-/.     

The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenografts

Summary An immunoconjugate composed of natural interferon (nIFN) bound in a noncleavable fashion to a monoclonal antibody (MoAb) recognizing a breast epithelial membrane mucin (Mc5) was used to treat xenografts of a human mammary carcinoma cell line (MCF-7) growing in nude mice. The immunoconjugate (nIFN/Mc5) was administered as 20 intralesional (i.l.) injections to 1 of 2 xenografts in each animal. It was found that nIFN/Mc5 produced a significant enhancement of the growth inhibitory actions of nIFN on the injected tumors. Further enhancement was obtained when nIFN or nIFN together with Mc5 (at a dose 10 times larger than that present in nIFN/Mc5) were added to the immunoconjugate. Biodistribution experiments showed that the uptake of¹²⁵I-nIFN/Mc5 by the tumors was greater and its elimination slower than for¹²⁵I-nIFN alone or conjugated to irrelevant mouse IgG₁. In addition, the immunoconjugate up-regulated the antigenic expression of a breast epithelial membrane mucin by the carcinoma cells, an up-regulation which was not significantly different from that produced by nIFN alone. The contralateral noninjected tumors exposed to systemic levels of the immunoconjugate showed an enhancement of antitumor effects, but to a lesser extent than the injected tumors. These findings suggest that the enhancement of the growth inhibitory action of the immunoconjugate was related to the specific binding of Mc5 which targeted the IFN to the carcinoma cells and impeded its elimination. It is likely that the targeting was favored by the IFN-mediated up-regulation of antigenic expression by the carcinoma cells, thereby producing a cascade of interrelated effects. The results of this study point out the feasibility and potential usefulness of IFN treatment by means of immunoconjugates as well as the worth of pursuing and improving this form of therapy.     

In vivo effects in mice produced by a 3′-branched homologue of α-2′-deoxythioguanosine

Summary Studies with a 3-branched chain homolog (-3-BCTGdR) of 2-deoxythioguanosine (-TGdR) showed that it did not prolong the survival of mice bearing the Mecca lymphosarcoma. Host toxicity was quite profound and resembled that seen with 6-thioguanine (6-TG). Evidence was obtained that this nucleoside derivative was not appreciably converted to 6-TG in the mouse. Mice treated with toxic doses of 6-TG or -3-BCTGdR were found to have very similar pathological changes. The granulocytes were eliminated from the peripheral blood, bone marrow was acellular, and some more limited damage was seen in the intestinal crypts. Experiments with radiosulfur-labeled drugs demonstrated that -3-BCTGdR was incorporated into the DNA of mouse bone marrow, predominantly in the chain-terminating position, with the result that shorter chains of DNA accumulated. The new homolog, unlike -TGdR, was phosphorylated in bone marrow as well as in tumor, and incorporated well into the DNA both of bone marrow and of the neoplastic cells. In devising other homologs attention must be given to the specificity of the kinases, i.e., to whether phosphorylation is superior in tumor cells or in the growing normal cells.Abbreviations used are 6-TG 6-thioguanine - ,-TGdR ,-2-deoxythioguanosine - -3-BCTGdR 6-thioguanine--2,3-dideoxy-3-(hydroxymethyl)-d-erythro-pentofuranose - ³H-TdR Tritium-labeled (methyl) thymidine     

Quantification of thrombospondin-1 secretion and expression of αvβ3 and α3β1 integrins and syndecan-1 as cell-surface receptors for thrombospondin-1 in malignant glioma cells

Malignant glioma cells secrete thrombospondin-1 (TSP-1) which participates in the motility of glioma cells, and binds to cell surface v3 and 31 integrins, and syndecan-1. This study evaluated the amount of TSP-1 secretion from malignant glioma cells, and the expression of v3 and 31 integrins, and syndecan-1. The amounts of TSP-1 in the supernatants from 10 malignant glioma cell lines and eight non-glioma malignant tumor cell lines were measured by enzyme-linked immunosorbent assay. Expression of v3 and 31 integrins, and syndecan-1 were examined by flow cytometry. The amounts of TSP-1 secreted by malignant glioma cells were 43 to 2431 ng/1 × 10⁶ cells/24 h (mean ± SD=626 ± 792). Seven of 10 glioma cell lines secreted more than 100 ng of TSP-1 and three of these cell lines secreted more than 1 g. Seven of eight non-glioma cell lines secreted less than 100 0ng of TSP-1. All glioma cell lines expressed 31 integrin and syndecan-1, and seven of 10 glioma cell lines expressed v3 integrin. Treatment of the glioma cell lines with TGF-2 did not change the expression of v3 integrin. These results suggest that malignant glioma cells secrete high levels of TSP-1, which may be important in the migration of glioma cells via interactions with v3 and 31 integrins, and syndecan-1.     

Effects of scheduling and ascites-associated macrophages on combined antiproliferative activity of alpha-2b interferon and gamma-interferon in a clonogenic assay

Summary Effects of combination treatment with human recombinant alpha-2b interferon (IFN-2b) and gamma interferon (IFN-) and sequencing of the combination on colony formation of human tumor cells were studied in a human tumor clonogenic assay (HTCA) with or without ascites-associated macrophages (AAM). Five different human tumor cell lines were studied. Three of the five cell lines (ovarian cancer cell line BG-1, cervical cancer cell line ME-180, and melanoma cell line SK-MEL 28) were sensitive to both IFNs.Cervical cancer cell line CaSki was sensitive to IFN-2b but resistant to IFN-. Endometrial cancer cell line HEC-1A was resistant to both IFNs. Synergistic interaction was observed in BG-1 and SK-MEL 28 with a combination of the IFNs. ME-180 did not exhibit a positive interaction, in spite of its sensitivity to each IFN. CaSki and HEC-1A also did not exhibit a positive combined interaction at clinically achievable concentrations. One sequential combination method (method 1: IFN-2b IFN gamma with a 24-h interval) resulted in a similar antitumor effect as the simultaneous combination. A reversed sequential method (method 2: IFN- IFN-2b with a 24-h interval) was less effective in three of the five cell lines. In BG-1, AAM enclosed in the lower layer markedly enhanced the antitumor effect of combined IFNs as well as each IFN alone. The antitumor effect with method 1 was significantly greater than that achieved with simultaneous combination or combination according to method 2 in the presence of AAM (P<0.01). These results suggest that (1) a synergistic antitumor effect of IFN-2b and IFN gamma is demonstrable in selected types of tumors, depending upon the sensitivity of each tumor cell line to both IFNs; (2) optimal scheduling for the direct antitumor effect of combined IFNs seems to be long-term exposure of cells to the IFN, the cells being treated with both IFNs either simultaneously or sequentially (IFN-2b preceding IFN-); and (3) AAM potentiate the antitumor effect of IFNs either alone or in combination. Finally, IFN-2b may have some priming effects for the indirect effect of IFN gamma mediated through AAM in certain tumor cells.     

Tumor necrosis factor-alpha induces interleukin-6 production via extracellular-regulated kinase 1 activation in breast cancer cells

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are frequently produced by breast cancer cells. These interleukins promote osteoclast formation and may mediate osteolysis at the site of breast cancer bone metastases. Transforming growth factor- (TGF-), tumor necrosis factor- (TNF-) and interleukin-1 (IL-1) up-regulate IL-6 and IL-11 production in a cytokine-dependent fashion in breast cancer cells, but very little is known about their intracellular signaling pathways in breast cancer cells. To study TGF-, TNF- and IL-1 regulation of IL-6 and IL-11 production in human MDA-MB-231 breast cancer cells, we established single cell clones stably expressing dominant negative (DN) forms of the mitogen-activated protein kinases p38 (p38/AF) or ERK1 (ERK1K71R). We show here, that while basal, TGF- and IL-1 induced IL-6 production was similar in parental cells and in pcDNA3 control, ERK1K71R and p38/AF clones, TNF- induced IL-6 production was blunted in the ERK1K71R clones. TGF- and IL-1, but not TNF-, induced IL-11 production in parental MDA-MB-231 cells. Similar findings were detected in clones stably expressing p38/AF and ERK1K71R, which did not change basal IL-11 production either. In conclusion, TNF- induced IL-6 production is mediated via ERK1 activation in MDA-MB-231 cells. These observations may be helpful in designing new anti-osteolytic therapies.     

Growth inhibitory effect of recombinant α and β interferon on human glioma cells

Summary Growth inhibitory activity of recombinant and interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant interferon showed slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant and interferon. The growth inhibitory activity of recombinant interferon on EFC-2 cells was not blocked by recombinant interferon, although recombinant and interferons shared same receptors on EFC-2 cells. Addition of DFMO (-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant interferon, and all were resistant to recombinant interferon. These results suggest differential sensitivity of EFC-2 cells to recombinant interferon, as well as a heterogeneous sensitivity to recombinant interferon among different glioma cell lines.     

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.     

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.     

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.     

Treatment of plasma cell neoplasm with recombinant leukocyte A interferon and human lymphoblastoid interferon

Summary Thisty cases of plasma cell neoplasms (24 multiple myeloma, one plasma cell leukemia, and three primary macroglobulinemia) were treated with two kinds of highly purified -interferons, recombinant human leukocyte interferon (rIFN-A) (16 cases) and human lymphoblastoid interferon (HLBI) (14 cases). Partial remission (PR) was obtained in two of 16 evaluable cases treated with rIFN-A and in two of 12 evaluable cases treated with HLBI. If minor response (MR) was included, responses were observed in seven (31.3%) and six (50%), respectively. Response (PR+MR) was noted in 38% of 21 previously treated patients and 71% of seven previously untreated patients. Side-effects were noted in more than two-thirds of the patients. They included fever, malaise, nausea/anorexia and myelosuppression. Thus, these two kinds of highly purified -interferon were effective in plama cell neoplasm, producing unequivocal response in 14.3% of the cases without unacceptable side-effects.     

Pharmacokinetics of an extended-release human interferon alpha-2b formulation

The in vivo half-life of human interferon alpha-2b (hIFN--2b) is relatively short, and frequent injections over prolonged periods are required for efficacy. An extended-release formulation of hIFN--2b (Depo/IFN) was created by encapsulation into a lipid-based drug-delivery system. The capture efficiency was 51%±13% and the release half-life in human plasma at 37°C was 16 days. The pharmacokinetics of Depo/IFN was compared with that of unencapsulated standard hIFN--2b (Std/IFN) in the peritoneal cavity of male BDF₁ mice. Depo/IFN exhibited a 13-fold longer intraperitoneal (i.p.) half-life as compared with Std/IFN (20 vs 1.5 h). The release of free hIFN--2b from Depo/IFN into the peritoneal cavity was slow and protracted, with a 10-fold lower peak concentration and a 13-fold longer apparent half-life being observed in comparison with Std/IFN. The areas under the curve of free hIFN--2b in the peritoneal cavity were comparable for Depo/IFN and Std/IFN. hIFN--2b was detectable in plasma only after the i.p. administration of Std/IFN. These data suggest the possibility that Depo/IFN may be useful as an extended-release formulation of hIFN--2b.     

Retinoid Receptors in Human Breast Carcinoma: Possible Modulators of in Situ Estrogen Metabolism

Retinoid receptors (retinoic acid (RARs) and retinoid X (RXRs) receptors) were immunolocalized in 32 human invasive ductal breast carcinomas. These findings were correlated with clinicopathological parameters to study their biological significance in breast carcinoma. Retinoid receptor immunoreactivity, except for RXR, was detected in the nuclei of carcinoma cells. Percentage of positive cases were RAR 81%, RAR; 6%, RAR; 28%, RXR; 81%, and RXR; 59%. A significant correlation was detected between RAR labeling index (LI), and RXR LI (r=0.667, p<0.001). Results from immunoblotting performed in three cases were consistent with those of immunohistochemistry. There was a significant correlation between RAR LI and 17-hydroxysteroid dehydrogenase (17-HSD) type 1 immunoreactivity (p<0.05). A significant correlation was also detected between RAR (r=0.413, p=0.019) or RXR (r=0.429, p=0.014) LI, and estrogen receptor (ER) LI. In T-47D breast cancer cells, which express RAR, RXR and ER, 17-HSD reductive activity increased 1.76-fold (p<0.001), five days following treatment with 10nM retinoic acid. These data suggest that retinoid receptors modulate various effects of retinoids, including estrogen metabolism in human breast carcinomas.     

Synergistic enhancement by interleukin-1 α of cisplatin-mediated antitumor activity in RIF-1 tumor-bearing C3H/HeJ mice

Administration of interleukin-1 (IL-1 ) plus certain cytotoxic drugs causes substantially greater clonogenic tumor-cell kill and tumor-regrowth delay than does treatment with either agent alone. IL-1 itself has little effect on tumor growth despite its ability to induce acute hemorrhagic necrosis, restrict tumor blood flow, and cause microvascular injury in a variety of murine model systems. To investigate further IL-1 's ability to enhance the antitumor activity of cytotoxic drugs, we initiated studies to examine the effect of IL-1 on cisplatin (cDDP)-mediated cytotoxicity using the RIF-1 tumor system. The antitumor activity of IL-1 and cDDP was quantitated through standard clonogenic tumor-cell survival assays, a tumor hemorrhagic necrosis assay and tumor-regrowth delay studies, with the interaction between IL-1 and cDDP being analyzed through median dose-effect. In vitro, IL-1 had no enhancing effect on the cDDP-mediated tumorcell kill. For examination of the in vivo efficacy of this regimen. RIF-1 tumor-bearing C3H/HeJ mice (14 days postimplantation) were treated concurrently with single i.p. injections of IL-1 and/or cDDP at various doses. The increased clonogenic tumor-cell kill obtained with IL-1 /cDDP was dose-dependent, with significant enhancement by IL-1 being observed (P<0.001), even at the lowest doses tested (2 mg/kg and 6 g/kg for cDDP and IL-1 , respectively), but it did not correlate with an increase in tumor hemorrhage. Using median dose-effect analysis, this interaction was determined to be strongly synergistic. When treated animals were monitored for long-term antitumor effects, combinations with IL-1 significantly increased the tumor-regrowth delay and decreased the fractional tumor volume (P<0.001). These results demonstrate that IL-1 synergistically enhances cDDP mediated in vivo antitumor activity and suggest that the combination of IL-1 and cDDP may have potential therapeutic application in the design of effective treatment modalities for cancer.Abbreviations IL-1 interleukin-1 - cDDP cis-diamminedichloroplatinum (cisplatin) - BRMs biological response modifiers - TNF tumor necrosis factor - IFN interferon - Fa traction affected - D_m or ED₅₀ drug concentration necessary to produce Fa=0.5 as compared with untreated controls - CI combination index; SF, surviving fraction - ANOVA oneway analysis of variance This work was supported in part by Public Health Service grant CA-48077 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, by the Mary Hillman Jennings Foundation, and by the Ramona DeSantis Cancer Research Fund     

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.     

Induction of tumour necrosis factor-α by single and repeated doses of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a low-molecular-weight biological response modifier scheduled for clinical evaluation, induced synthesis of tumour necrosis factor- (TNF-) in serum of mice, with maximal activity being observed at 2–3 h after administration. At a dose of 27.5 mg/kg, DMXAA induced similar TNF- concentrations as did flavone-8-acetic acid given at its maximum tolerated dose (MTD; 330 mg/kg), whereas 8-methylxanthenone-4-acetic acid, which has no antitumour activity, did not induce serum TNF- at its MTD (440 mg/kg). The dependence of schedule on TNF- induction was studied by giving DMXAA to mice in two doses of 27.5 mg/kg each separated by different intervals. An interval of 0 (i.e 55 mg/kg given in a single dose) produced a TNF- concentration 9-fold that produced hy a single dose of 27.5 mg/kg. This dose, although higher than the MTD of 30 mg/kg, did not affect the health of mice at the time of assay (3 h). An interval of 1 day produced very low levels of serum TNF- after the second injection. An interval of 3 days produced high levels of serum TNF- after the second injection (9-fold that detected in mice receiving 27.5 mg/kg in a single dose) but no long-term toxicity, whereas an interval of 7 days produced an intermediate response. Thus, the first dose can either potentiate or suppress the TNF- response to a second dose. Mice with advanced subcutaneous colon 38 tumours were treated either with a single dose of DMXAA (27.5 mg/kg) or with a divided dose (two doses of 27.5 mg/kg given 3 days apart). Both the cure rate and the tumour-growth delay were enhanced by the divided-dose schedule. The results are relevant to the design of clinical administration schedules of DMXAA and emphasise the importance of TNF- induction in the antitumour response.This study was supported by the Auckland Division of the Cancer Society of New Zealand and by the Health Research Council of New Zealand     

Doxorubicin-induced hair loss in the Angora rabbit: a study of treatments to protect against the hair loss

Summary An animal model for anticancer drug-induced hair loss has been developed using the Angora rabbit given i.v. doxorubicin, 2 mg/kg, twice weekly for 3 weeks. There was a 167% increase in the weight of hair collected by grooming between weeks 2 and 5, and a 72% inhibition of new hair growth at week 6 compared with non-treated animals. The hairs that grew in the doxorubicin treated rabbits did so at the same rate as in non-treated rabbits and appeared normal by light microscopy. Topical application of dimethylsulfoxide (DMSO), of 10% -tocopherol in DMSO, of 0.5% naphthazoline hydrochloride in DMSO, of 0.1% fluocinolone acetonide in a propylene glycol base and local hypothermia did not provide any protection against doxorubicin-induced hair loss. Angora rabbits fed an -tocopherol-deficient diet for 6 weeks showed decreased hair growth compared with animals fed a normal diet or a diet supplemented with 100 mg -tocopherol acetate twice a week for 6 weeks. Some rabbits fed the -tocopherol-deficient diet died when given doxorubicin. Rabbits fed the -tocopherol-supplemented diet showed evidence of protection against doxorubicin-dependent inhibition of new hair growth.     

Overexpression of the p73 gene is a novel finding in high-risk B-cell chronic lymphocytic leukemia

The p73 protein shares structural and functional similarities with thetumour-suppressor p53, but its role in neoplastic transformation is unknown.Alternative splicing leads to the expression of at least nine p73 C-terminalmRNA splice variants (, , , , , , ,1, ). In this survey, we analyse the expression of p73 byreal-time quantitative RT–PCR, its known C-terminal variants with anRT–PCR-Southern technique and by Western blot in samples of 51 patientswith B-CLL, normal B lymphocytes from eight individuals, and fivehaematopoetic cell lines. p73 protein expression positively correlatedwith higher risk B-CLL stages (P = 0.046). Total p73 mRNAexpression was higher (P = 0.01) and p73 protein morefrequently detected (P = 0.008) in B-CLL compared with normalCD19+–B-lymphocytes. p73 C-terminal mRNA variants were expressed bothin B-CLL and in normal B-lymphocytes, but their expression was biased sincethe (P = 0.041), the (P <0.001), and the variant (P = 0.033) prevailed in normalB-lymphocytes. In summary, we conclude that the accumulation of p73, theexpression pattern of particular p73 variants and its link to progression mayplay a distinct role in the molecular pathology B-CLL.     

Glucocorticoids and androgens up-regulate the Zn-α2-glycoprotein messenger RNA in human breast cancer cells

Summary We have studied the hormonal regulation of the gene encoding Zn-₂-glycoprotein (Zn-₂-gp), a human protein with a high degree of amino acid sequence similarity to class I histocompatibility antigens that is produced by a specific subset of breast carcinomas. Northern blot analysis revealed that dexamethasone and 5-dihydrotestosterone strongly induced the accumulation of Zn-₂-gp mRNA in T-47D human breast cancer cells. Furthermore, the effect of these two hormones was shown to be additive, since the combination of both hormones produced a stimulation of Zn-₂-gp mRNA of at least 3-fold over that produced by either hormone alone. By contrast, the addition of 5-dihydrotestosterone, 17-estradiol, or progesterone failed to induce the expression of Zn-₂-gp. The stimulatory effect of glucocorticoids and androgens on Zn-₂-gp expression was produced in a time and dose dependent manner, without significantly affecting the cell proliferation rate. A time-course study demonstrated that the induction of Zn-₂-gp mRNA by androgens and glucocorticoids reached a level of 4 or 3.2-fold over the untreated control after seven days of incubation in the presence of a 10^–7 M concentration of 5-dihydrotestosterone or dexamethasone, respectively. A dose-response study showed that as little as 10^–11 M of 5-dihydrotestosterone or dexamethasone produced an accumulation of Zn-₂-gp mRNA of 2.4 or 2.1-fold over the control, respectively. On the basis of these results, we propose that Zn-₂-gp may be useful as a biochemical marker of breast carcinomas with a specific pattern of hormone responsiveness in whose development glucocorticoids and/or androgens may play a significant role.