Selective interactions of verapamil with anthraquinones in adriamycin-sensitive and-resistant murine and human tumour cell lines in vitro

Summary Enhancement of the cytotoxicity of adriamycin (ADR) by addition of verpamil (VPM) was selective and, in part, concentration-dependent. In an ADR-resistant murine L5178Y lymphoma subline sensitivity was improved with ADR and 1 M VPM, whereas the cytotoxic effects of mitoxantrone or esorubicin were not affected by VPM. In human ovarian SK-OV-3 tumour cells ADR cytotoxicity was only enhanced by 6.6 M VPM and there was no selective advantage against an ADR-resistant subline. Circumvention of ADR resistance by VPM is not a universal phenomenon, appearing least effective against sublines expressing relatively low orders of resistance, which may be those most commonly encountered clinically.     

Cytokine‐regulated urokinase‐type‐plasminogen‐activator (uPA) production by human breast fibroblasts in vitro

It has been shown that, in breast stroma, urokinasetype plasminogen activator (uPA) mRNA is predominantly expressed by myofibroblasts located at the invasive areas of the tumor. To examine which factors present in a tumor environment are candidates responsible for the induction of these uPAproducing myofibroblasts, we studied in vitro the capacity of a paired panel of normal and tumorderived human breast fibroblasts to produce uPA protein and the myofibroblast marker smoothmuscleactin (SMA) in response to various cytokines implicated in the process of tissueremodeling during malignant transformation.We found that fibroblasts produced increased amounts of uPA protein after exposure to aFGF, bFGF, EGF, PDGFBB, and IFN, were unaffected in this respect by IL6, MCSF, GMCSF and Oncostatin M, and produced decreased amounts of uPA protein after exposure to IL1, TNF, IGFI, and IGFII. None of these cytokines were able to induce a striking increase in the fraction of SMApositive fibroblasts. On the other hand, 25pM TGF₁ increased the fraction of SMApositive fibroblasts 5fold in both normal and tumortissuederived fibroblasts. Nonetheless, the normalderived fibroblasts were unaffected in their uPAproducing capacity by TGF₁, and the tumorderived fibroblasts produced decreased amounts of uPA protein after exposure to this cytokine, implying that at least in vitro the myofibroblast phenotype is not a prerequisite for the production of uPA by human breast fibroblasts. In addition, we established that the basaluPAproduction of both normal and tumorderived fibroblasts was increased by autocrinely produced bFGFlike activity, and that the basaluPAproduction of at least the normalderived fibroblasts was decreased by autocrinely produced IGFlike activity.Altogether, our data suggest an active role for fibroblasts in the process of uPAdirected breast tumor proteolysis.     

Effect of irradiation on transforming growth factor-β secretion by malignant glioma cells

Glioblastoma cells secrete transforming growth factor- (TGF-), whichhas a variety of immunosuppressive properties. We investigatedthe effect of irradiation TGF- secretion by malignantglioma cells. Three malignant glioma cell lines (T98G,A172, KG-1-C) were cultured and irradiated using 10and 50 Gy Linac radiation. After further culturefor 36 hours in serum-free culture medium, thesupernatants were collected. The TGF- activity in theculture supernatants was determined using a specific bioassay.The levels of the active form and totalTGF- in the supernatants from irradiated malignant gliomacells decreased compared to those from un-irradiated cells.However, since irradiation inhibited the growth of tumorcells, the amount of TGF- secretion per cellin irradiated cells tended to increase after irradiation.These results suggest that malignant glioma cells canstill secrete TGF- and activate latent TGF- evenafter large dose irradiation, despite the inhibition oftumor growth.     

Neuroblastoma and parental occupation

Objectives: We evaluated parental occupation and the risk of neuroblastoma using data from a large case–control study conducted by the Children's Cancer Group and the Pediatric Oncology Group.Methods: We compared the distribution of 73 paternal and 57 maternal occupational groups among 504 newly diagnosed cases of neuroblastoma and individually matched controls obtained by telephone random digit dialing in the United States and Canada.Results: An increased risk of neuroblastoma was found for fathers employed as broadcast, telephone and dispatch operators (odds ratio [OR]=6.1; 95% confidence interval [CI]=0.7–50.9), electrical power installers and power plant operators (OR=2.7; CI=0.9–8.1), landscapers and groundskeepers (OR=2.3; CI=1.0–5.2), and painters (OR=2.1; CI=0.9–4.8). Elevated odds ratios were found for mothers employed as farmers and farm workers (OR=2.2; CI=0.6–8.8), florists and garden store workers (OR=2.4; CI=0.6–9.9), hairdressers and barbers (OR=2.8; CI=1.2–6.3), electric power installers and power plant operators, and sailors, fishers, and railroad workers. No increase in risk was found for other paternal occupations previously associated, including electricians, electrical equipment assemblers and repairers (OR=1.1; CI=0.6–2.0), or welders (OR=0.5; CI=0.1–1.6).Conclusion: The study reinforced some prior evidence of increased risks in electrical, farming and gardening, and painting occupations, but failed to confirm other previously reported associations. Further analyses of exposure to electromagnetic fields, metals, solvents, and pesticides are currently under way.     

Reversal of estrogen‐resistance in murine mammary adenocarcinomas

From mouse mammary progestindependent (PD) adenocarcinomas induced with medroxyprogesterone acetate (MPA) we developed several in vivo lines that are maintained by subcutaneous syngeneic passages in MPAtreated mice and express estrogen (ER) and progesterone receptors (PR). Although most lines remained PD, with time some progestinindependent (PI) variants arose. Both PD and PI tumors regress with estrogen treatment although estrogenresistant variants may also arise. The object of this paper was to investigate the reversibility of estrogenresistance and its possible relation with hormone receptor downregulation. Tumor regression was induced in a progestinindependent tumor line (BET) by treatment with a 5mg 17estradiol (E2) silastic pellet. One of the tumors started to grow disclosing an estrogenresistant pattern of growth. This tumor line (BETR) was transplanted into E2treated and untreated animals (n= 4–6), selecting for the next passage tumors growing in treated animals. Seven new sublines were obtained at different passages by selecting for the next passage the tumors that had grown in untreated mice (BETRaBETRg), until no tumors grew in E2treated mice. ER and PR were measured by a ligandbinding, dextrancoated charcoal method using a single saturating point. From the seven sublines initiated, the first four proved to be reversible after 3–6 generation transplantation and the last three did not revert. A difference in PR expression between BET and BETR (p < 0.05) was registered, but it did not correlate with the specific hormone behavior since two reverted lines had a pattern similar to that of BET and the other two were similar to BETR. The expression of PR was higher in E2treated mice (p < 0.05) and highly variable in the parental line. This led us to study the expression of PR at different stages of the estrous cycle. Higher levels of PR were observed in proestrous, estrous, and metestrous (p < 0.05) than in diestrous, and undetectable levels were found in ovariectomized mice. No differences in ER expression were detected during the estrous cycle. It can be concluded that under certain experimental conditions, estrogenresistance is a reversible phenomenon. The experimental manipulation of hormone resistance may help develop strategies to modify the response to antihormones in humans.     

Suppression of Cell Invasion on Human Malignant Glioma Cell Lines by a Novel Matrix-metalloproteinase Inhibitor SI-27: In Vitro Study

Matrix metalloproteinase (MMP) has come to be highlighted by its close relation to the cell invasion of gliomas. Suppression of MMP activity in malignant glioma cells would be meriting to local delivery of genes or chemotherapeutic agents. In this study, we employed a novel MMP inhibitor, SI-27 to investigate inhibition of cell invasiveness in human malignant glioma cell lines, U87MG, U251MG, and U373MG. We evaluated with zymogram, reverse zymogram, and cell invasion assay after exposure of SI-27 for 24h followed by preliminary MTT assay to find non-cytotoxic dose range, 51050100g/ml compared with non-treatment group as the control. Common to three glioma cell lines, zymogram disclosed that expressions of MMP-2 and -9 were suppressed in a dose-dependent fashion, meanwhile those of tissue inhibitor of MMP (TIMMP) in reverse zymogram were not. The numbers of invading cells through Boyden chamber were significantly reduced in a dose-dependent manner, while those with 5g/ml were not diminished common to those three lines. In conclusion, dose concentration ranging 10–100g/ml of SI-27 inhibited MMP-2 and -9 mediated cell invasiveness in malignant glioma cell lines. This is the first report for chemotherapeutic effect of SI-27 on glioma cells.     

Treatment of advanced breast cancer with gemcitabine and vinorelbine plus human granulocyte colony‐stimulating factor

Purpose. A phase II trial was performed to investigate the efficacy and tolerance of gemcitabine, vinorelbine, and recombinant human granulocyte colonystimulating factor (GCSF) in advanced breast cancer. Patients and methods. Between April 96 and August 97, 60 patients entered this trial. Fortyfive patients were previously untreated and 15 patients had failed previous palliative chemotherapy with (n = 10) or without anthracyclines (n = 5). Therapy consisted of gemcitabine 1000mg/m² on days 1 + 15 + 21 and vinorelbine 40mg/m² on days 1 + 21, both diluted in 250ml saline and infused over 30min. GCSF was administered at 5g/kg/day subcutaneously from days 2–6 and 22–26. Courses were repeated every 5 weeks. Treatment was continued in case of response or stable disease until a total of six courses. Results. The overall response rate was 55.5% for patients who had not received prior palliative chemotherapy (95% confidence interval, 40%–70.3%), including 5 CR (11.1%) and 20 PR (44.4%) 12 patients (27%) had stable disease (SD), and 8 (18%) progressed. Secondline treatment with this regimen resulted in 6/15 (40%) objective remissions, 5 had SD, and 4 PD. The median time to progression was 9.5 months (range, 1.5–28) in previously untreated patients, and 7.0 months (range, 2–23) in those who had failed prior chemotherapy. After a median followup time of 15 months, 44 patients (73%) are still alive with metastatic disease. Myelosuppression was commonly observed, though WHO 3 and 4 neutropenia occured in only 9 (l5%) and 2 patients (3%), and was never complicated by septicaemia; grade 3 anemia was noted in 2 patients. Severe (WHO grade 3) nonhematologic toxicity was rarely observed, and included nausea/emesis in 3 and constipation in 2 patients. Conclusions. Our data suggest that gemcitabine and vinorelbine plus GCSF is an effective and tolerable first as well as secondline combination regimen for treatment of advanced breast cancer.     

Accuracy of mammography and echography versus clinical palpation in the assessment of response to primary chemotherapy in breast cancer patients with operable disease

The response to primary chemotherapy is an important prognostic factor in patients with non metastatic breast cancer. In this study we compared the assessment of response performed by clinical palpation to that performed by echography and mammography in 141 out of 157 consecutive breast cancer patients (T2-4, N0-1, M0) submitted to primary chemotherapy. A low relationship was recorded between tumor size assessed clinically and that evaluated by either mammography: Spearman R=0.38 or echography: R=0.24, while a greater correlation was found between the tumor dimension obtained by the two imaging techniques (R=0.62). According to the WHO criteria, the grade of response of breast cancer to primary chemotherapy, showed by mammography and echography, was less marked than the grade of response seen at clinical examination. Residual tumor size assessed clinically depicted a stronger correlation with pathological findings (R=0.68) than the residual disease assessed by echography (R=0.29) and mammography (R=0.33). Post-chemotherapy histology evaluation revealed pathological complete response in three cases (2.1%). Two of these cases were judged as complete responders by clinical palpation but only one was recognized by mammography, and none by echography. Clinical response, but not the response obtained by the two imaging techniques, was a significant predictor for longer disease free survival (p=0.04). To conclude, physical examination measurements remain the method of choice in evaluating preoperatively the disease response in trials of primary chemotherapy. Prediction of pathological outcome is not improved by echography and mammography.     

Biological and clinical evaluation of Lanreotide (BIM 23014), a somatostatin analogue,in the treatment of advanced breast cancer

Summary Recently, compounds having pure antiestrogenic activity have become available. In this study, we examined the activity of the new steroidal antiestrogen EM-170 (N-n-butyl, N-methyl-11-(16-chloro-3,17-dihydroxy-estra-1,3,5-(10)-trien-7-yl) undecanamide) on the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma stimulated by treatment with estrone (E₁), a steroid known to play an important role as precursor of 17-estradiol (E₂), especially in postmenopausal women. Twenty-five days after ovariectomy (OVX), tumor volume in control OVX animals decreased to 51.4 ± 11% of the initial volume; treatment with E₁, administered by Silastic implants, stimulated tumor growth to 179 ± 21%. Treatment with the antiestrogen EM-170 at a dose of 200 µg (twice daily) not only completely reversed the stimulatory effect of E₁, but also inhibited tumor growth to 30.5 ± 9.6%, an effect that is 41% (P < 0.01 vs OVX control) greater than that of ovariectomy alone. At a relatively low dose of 40 µg (twice daily), 20 days of treatment with EM-170 reversed by 55% the stimulatory effect of E₁ (1.0 µg, subcutaneously, twice daily) on tumor growth in OVX animals. On the other hand, the antiestrogen also induced a significant inhibitory effect on 17-hydroxysteroid dehydrogenase (17-HSD) activity in the DMBA-induced mammary tumors, an effect that is in agreement with the marked reduction caused by the same treatment on tumor estradiol (E₂) levels in E₁-treated OVX animals. The present data show that the new steroidal antiestrogen EM-170 exerts a potent inhibitory effectin vivo on E₁-stimulated growth of DMBA-induced mammary tumors, an effect that is probably mediated by both its antiestrogenic activity at the receptor level and its inhibitory effect on 17-HSD, thus inhibiting local E₂ formation and facilitating the action of the antiestrogen at the receptor level.     

Glycogen‐rich carcinomas of the breast display unique characteristics with respect to proliferation and the frequency of oligonucleosomal fragments

We determined the proliferation rate and apoptotic activity of glycogenrich carcinomas of the breast as opposed to nonclear cell tumors by means of MIB1 immunohistochemistry and in situ detection of oligonucleosomal fragments (TUNEL reaction). The retrospective biopsy series included six invasive clear cell carcinomas of the glycogenrich type as well as 15 randomly selected cases of invasive ductal carcinoma without evidence of glycogen storage. Three patients in the clear cell group and seven patients in the control cohort developed lymphnode metastasis. The MIB1 labeling index of glycogenrich carcinomas averaged 9.05%, while that of the controls was 30.03%. Apoptotic nuclei were present in a mean of 1.26% of glycogenrich carcinoma cells. The control tumors exhibited an average apoptotic frequency of 5.85%. Tumor size, hormone receptor status, and presence or absence of lymph node involvement were found not to correlate with either proliferation or apoptosis. We conclude that glycogenrich breast carcinomas are characterized by a peculiar low proliferationlow apoptosis cell kinetic profile. The aggressive clinical behavior of these neoplasms may possibly be accounted for by an ineffective apoptotic elimination of otherwise slowly proliferating tumor cells.     

Quantitative changes in cytological molecular markers during primary medical treatment of breast cancer: A pilot study

Aim: To quantify the changes in biological molecular markers during primary medical treatment in patients with operable breast cancer and to assess their possible relationship with response to treatment.Methods: The treatment group consisted of 31 patients with operable breast carcinomas, median age 57 years (range 41–67), treated with four 3weekly cycles of chemotherapy with Mitoxantrone, methotrexate (± mitomycin C), and tamoxifen before surgery. Fine needle aspiration (FNA) was used to obtain samples from patients prior to and at 10 or 21 days posttreatment. The following molecular markers were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Bcl2, and Ki67 measured by immunocytochemistry, and ploidy and Sphase fraction (SPF) by flow cytometry. To evaluate the reproducibility of the technique, repeat FNA was performed in a separate nontreatment control group of 20 patients and the same molecular markers assessed, two weeks after the first sample with no intervening treatment.Results: The nontreatment control group showed a high reproducibility for the measurement of molecular markers from repeat FNA. In the treatment group there was a nonsignificant reduction in SPF and a significant reduction (p = 0.005) in Ki67. Patients who responded to neoadjuvant therapy were more likely to have a reduction in these two markers than those who failed to respond. Similarly, a reduction in ER scores was observed between the first and second samples (p = 0.04). For PgR, the change between the first and second samples was not significant although there was a significant difference between responders and nonresponders (p = 0.03). All nine patients with an increase in PgR were responders. No significant changes in p53 or Bcl2 were observed during treatment.Conclusion: Molecular markers can be adequately measured from FNA samples prior to and during neoadjuvant therapy. Changes in cellular proliferation and hormone receptors have been shown that may be related to tumour response. These relationships should be assessed in a larger cohort of patients.     

Progression of Premalignant MCF10AT Generates Heterogeneous Malignant Variants with Characteristic Histologic Types and Immunohistochemical Markers

The purpose of this study was to evaluate the pain experience of women during mammography for breast cancer screening. Possible associations with personal and medical history, sociodemographics and/or situational factors were studied. It was also investigated whether this pain influenced the intention to return for future breast cancer screening. In the Netherlands, women between 50–75 years are invited for screening every two years. A total of 1200 participants were asked to fill up a questionnaire. The response rate was 79.5% (n=954), and 945 questionnaires contained adequate information for analyses. A total of 689 women (72.9%) described mammography as mild to severely painful. In this group, compared to the group that reported no pain, the following factors occurred significantly more often: sensitive breasts (P=0.001), family history of breast diseases (P=0.017), expected pain based on former mammography (P=0.001), high education (P=0.008), anxiety (P=0.001), breast sensitivity in last three days (P=0.001), insufficient attention of technologist (P=0.001). Other factors like age, hormonal status, breast size and hormone use were not associated with the pain experienced. Thirty-two women (3.3%) indicated that they would not attend further screening, 25 (2.6%) reported that the pain might deter them, six women (0.6%) had other reasons, one woman (0.1%) was sure not to come because of severe pain. In conclusion, a large majority of women attending breast cancer screening describes mammography as painful (72.9%). Factors associated with pain were described. Relatively few women (2.7%) indicated that the pain might deter them from future mammography. Recommendations are given to reduce the pain experienced during screening mammography.     

Multiple actions of synthetic ‘progestins’ on the growth of ZR-75-1 human breast cancer cells: Anin vitro model for the simultaneous assay of androgen,progestin, estrogen,and glucocorticoid agonistic and antagonistic activities of steroids

This study was designed to assess the multiple steroid receptor mediated activities of a series of synthetic progestins on breast cancer cell growth, using the human ZR-75-1 cell line which possesses functional estrogen (ER), androgen (AR), and glucocorticoid (GR) receptors as well as progesterone (PgR) receptors. Four 17-hydroxyprogesterone derivatives (chlormadinone acetate, CMA; cyproterone acetate, CPA; medroxyprogesterone acetate, MPA; and megestrol acetate, MGA) and two 19-nortestosterone derivatives (norethindrone, NRE, and norgestrel, NRG) were thus investigated.Based on the requirement of estrogens for PgR-mediated antiproliferative effects and the reversal of PgR-mediated action by insulin, it was found that although all progestins could inhibit ZR-75-1 cell growth through the PgR at low concentrations, the relative contribution of this receptor in cell growth control is highly variable between compounds. The quantitative importance of PgR-mediated inhibition of cell proliferation was inversely related to the amplitude of the androgenic effects induced by the compounds, the AR-mediated effects increasing in the order CPA < MGA < CMA < NRE < NRG < MPA. The specificity of these androgenic effects is further supported by their reversal upon addition of the antiandrogen hydroxyflutamide. In addition, the 17-hydroxyprogesterone derivatives, but not the 19-nortestosterone derivatives, had glucocorticoid activities at high (micromolar) concentrations, as shown by reversal of growth inhibition by the antagonist RU486 in the presence of saturating concentrations of 5-dihydrotestosterone. All progestins tested, except MPA and NRE, also had some antiglucocorticoid activity, NRG being the most potent in this respect. Finally, NRE and NRG exerted a marked mitogenic effect in estrogen-free medium which was clearly mediated through the ER as shown by the competitive reversal of their action by the steroidal antiestrogen EM-139.The present results show that growth measurements of the human breast cancer cells ZR-75-1 permit, with the appropriate steroid additions, the assay of progestin, androgen, estrogen, and glucocorticoid agonistic as vell as antagonistic activities of test compounds. The present study shows, somewhat surprisingly, that while the AR is almost completely responsible for the action of MPA at low concentrations, the majority of the action of NRE, NRG, and MGA is also exerted through AR, while the androgenic action of CPA plays a lower role in the growth inhibition induced by this compound. Such a model should be of great help in designing more specific steroid drugs and in better understanding the role of the different steroid classes which can be used to control the growth of hormone-sensitive cancer. The present data also indicate that progestin is an inappropriate name for MPA, NRE, NRG, MGA, CMA, and CPA, which all possess other and sometimes more potent steroidal activites than those related to interaction with the progesterone receptor.Abbreviations CMA chlormadinone acetate [17-acetoxy-6-chloropregna-4, 6-dien-3, 20-dione] - CPA cyproterone acetate [17-acetoxy-6-chloro-1,2-methylene-pregna-4, 6-dien-3, 20-dione] - DEX dexamethasone [9-fluoro-11, 17, 21-trihydroxy-16-methyl-pregna-1, 4-dien-3, 20-dione] - DHT 5-dihydrotestosterone [17-hydroxy-5-androstan-3-one] - E₂ estradiol [estra-1, 3, 5 (10)-trien-3, 17-diol] - EM 139 [N-n-butyl-N-methyl-11-(16-chloro-3, 17-dihydroxyestra-1, 3, 5 (10)-triene-7-yl) undecanamide] - MGA megestrol acetate [17-acetoxy-6-methylpregna-4, 6-dien-3, 20-dionel] - MPA medroxyprogesterone acetate [17-acetoxy-6-methylpregn-4-en-3, 20-dione] - NRE norethindrone [17-hydroxy-19-nor-17-pregn-4-en-20-yn-3-one] - NRG norgestrel [13-ethyl-17-hydroxy-18, 19-dinor-17-pregn-4-en-20-yn-3-one] - OHF hydroxyflutamide (SCH 16423) [, , -trifluoro-2-methyl-4-nitro-m-lactotoluidide] - R1881 methyltrienolone [17-hydroxy-17-methyl estra-4, 9, 11-trien-3-one] - R5020 promegestone [17, 21-dimethyl-19-norpregna-4, 9-dien-3, 20-dione] - RU486 [17-hydroxy-11-(4-dimethylaminophenyl)-17-(1-propynyl)-estra-4, 9-dien-3-one] - triamcinolone acetonide [9-fluoro-11, 21-dihydroxy-16, 17(1-methylethylidenebis oxy) pregna-1, 4-dien-3, 20-dione]     

Expression of LFA-1/ICAM-1 in CNS lymphomas: possible mechanism for lymphoma homing into the brain

We examined a possible role for the adhesion molecules LFA-1 and ICAM-1 in localizing central nervous system non-Hodgkin's lymphomas (CNS-NHLs) to the brain. Fresh frozen sections from 12 monoclonal CNS NHLs (11 primary, one secondary) were stained with monoclonal antibodies to LFA-1 chain (CD11a), chain (CD18) and, ICAM-1 (CD54). Additional staining made use of rat monoclonal antibodies to the human and mouse high endothelial venule antigens HECA 452 and MECA 79 and mouse ICAM-1. The expression of these same molecules was also studied in mice with severe combined immunodeficiency (SCID) mice, bearing intracranial human lymphoblastoid cells.Eleven of the CNS-NHL tumors expressed LFA-1 (one strongly, one intermediate, nine weakly). Nine of the tumors weakly expressed LFA-1.. Nine of twelve tumors weakly expressed ICAM-1. In six of seven tumors definite blood vessels stained for ICAM-l. Non-tumor brain from two patients and non-tumor cerebral blood vessels showed no staining with CD11a, CD18 or CD54 antibodies. Strong expression of LFA- and LFA- as well as ICAM-1 was noted in human lymphoblastoid cells (LCLs)/SCID mouse CNS lymphomas. Tumor blood vessels in these mice stained for mouse ICAM-1. Normal SCID mouse brains showed no staining with CDII, CD18, CD54 or mouse ICAM-1 antibodies. Human, human/mouse CNS lymphomas, normal human, and mouse brains showed no staining with either HECA 452 or MECA 79.Our data suggests that CNS lymphomas express LFA-1, LFA-1 and to a lesser degree ICAM-1 antigen, while tumor blood vessel endothelium expresses ICAM-1. LFA-1-LFA-1/ICAM-1 interaction may play a role in large cell lymphoma homing and persistence in the brain.     

Clinicopathological characteristics of breast carcinomas with allelic loss in the p73

p73, a new member of the p53 family, has been mapped to chromosome 1p36, a region where loss of heterozygosity (LOH) is frequently observed in primary human tumors. Allelic loss studies involving the 1parm in breast carcinomas offer rates ranging from 13% to 75%, depending on the genetic interval being studied. We investigated LOH in an intragenic microsatellite marker, and those centromerically flanking the p73 gene, at 1p36, and their correlations with patient age and 10 pathologic parameters in a series of 193 breast carcinomas. The LOH analysis was performed by amplifying DNA by PCR, using five markers of the 1p36 region (p73P1, D1S2694, D1S214, D1S2666 and D1S450). LOH was found in at least one of these markers in 27% of tumors. When we established the comparison between tumors with and without LOH and the distribution of the 10 pathologic parameters considered, we observed statistically significant differences in association with higher histologic grade (p=0.02), more advanced pathological stage (p=0.02), peritumoral vessel involvement (p=0.04) and poorly differentiated carcinomas (p=0.01), as well as in tumors that concomitantly exhibited lymph node metastases, peritumoral vessel involvement and absence of steroid receptors (p=0.02). These data suggest that LOH in the p73 region could be pathogenically related to breast cancer and possibly to a poor tumor prognosis.     

Tissue inhibitor of metalloprotease (TIMP)‐1 and proliferative behaviour of clonal breast cancer cells

In the present paper, we have examined whether human tissue inhibitor of metalloprotease1 (hTIMP1) is able to exert a growth factorlike effect on two clonal cell lines (BC3A and BC61), isolated from a parental line of human breast carcinoma cells (8701BC), and endowed with different growth and invasive behaviour in vitro and in nude mouse. The data obtained indicate that only the more tumorigenic clonal cell line (BC61) is responsive to hTIMP1 treatment by increasing its proliferative rate in a dosedependent manner. It was also found that BC61 cells selectively express a transmembrane protein of about 80kDa able to bind hTIMP1 in vitro and in vivo with high affinity (K_d of 0.07 ± 0.004 nM), and that treatment of BC61 cells with a proliferationpromoting concentration of hTIMP1 is able to stimulate tyrosinetargeted phosphorylation. The cumulative results obtained strongly support the hypothesis that hTIMP1, classically regarded as a collagenase inhibitor, may be a crucial element of the extracellular signalling network during breast cancer development by controlling cell growth phenotype in autocrine and paracrine manner, and that intratumoural heterogeneity for the biological response to TIMP1 may exist within the composite cell population of the primary tumour site.     

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-/.     

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     

High complete pathological response in locally advanced breast cancer using paclitaxel and cisplatin

Background.In an earlier study, we have demonstrated a high response rate in metastatic breast cancer using paclitaxel (P) and cisplatin (C). A phase II study using the same regimen (PC) has been conducted in locally advanced breast cancer (LABC). Methods.A total of 72 consecutive patients with non-inflammatory LABC (T24cm, T3 or T4, N0–N2, M0). Patients were scheduled to receive 3–4 cycles of the neoadjuvant PC (paclitaxel 135mg/m² and cisplatin 75mg/m² on day 1) every 21 days. Patients were then subjected to surgery and subsequently received 6 cycles of FAC (5-fluorouracil 500mg/m², doxorubicin 50mg/m², and cyclophosphamide 500mg/m²) or 4 cycles of AC (doxorubicin 60mg/m², and cyclophosphamide 600mg/m²). Patients then received radiation therapy, and those with hormone receptor positive tumors were given adjuvant tamoxifen intended for 5 years. Results.The median age was 39 years (range, 24–78). Clinically, 7%, 58%, and 35% of patients had T24cm, T3, and T4, respectively. Disease stage at diagnosis was IIB (33%), IIIA (27%), and IIIB (40%). Complete and partial clinical response to PC was demonstrated in 13 (18%), and 52 (72%) patients, respectively. Of those patients with evaluable pathologic response (68 patients), complete pathologic response (pCR) was achieved in 15 (22%) patients. At a median follow-up of 22 (±3.5) months, 58 (81%) were alive with no recurrence, nine (12%) were alive with evidence of disease, and five (7%) were dead. None of the patients achieving pCR has developed any relapse. The median overall survival has not been reached for all 72 patients with a projected 3-year survival (±SE) of 90% (±4%). The median progression-free survival (PFS) was 42.1 (±4.8) months with a projected PFS of 74%±7% at 3-years (for 68 patients). Conclusions.PC regimen in LABC produced a high pCR. The contribution of the other added modalities to survival could not be assessed.