Biomedical subjects
Publications and source records attributed to A Alama.
A total of 22 relapsed or refractory ovarian cancer patients were treated with ifosfamide-containing polychemotherapy. Kinetic analyses were done to evaluate the tumor-cell-recruiting potential of the alkylating agent. Our study shows that ifosfamide can enhance tumor proliferative activity in pretreated ovarian cancers.
After standard management of stage III-IV ovarian cancer patients by surgical reduction of tumor mass and subsequent cisplatin-based combination chemotherapy, eradication of residual intraabdominal disease remains a major clinical problem. In an effort to increase response to therapy without adding marrow toxicity, after laparotomy, 21 stage III-IV ovarian cancer patients were treated with systemic chemotherapy comprising cisplatin, doxorubicin, and cyclophosphamide (PAC) on Day 1 followed by intraperitoneal (ip) cytosine arabinoside (Ara-C) on either Day 8 or 14, every 28 days. Ara-C, an S-phase-specific drug, was administered ip to exploit the pharmacologic advantage of an ip regimen at a time when a possible PAC-induced recruitment of cells into the proliferative pool could further maximize cell kill. Kinetic features of ovarian neoplastic cells recovered from peritoneal washings were monitored during treatment by measurement of the thymidine labeling index (TLI): data from four patients indicate that there is an increase in proliferating cells on Days 8 and 14 after PAC treatment. Toxicity of treatment was acceptable. Although 95% of evaluable patients had more than 2 cm of residual disease, response was observed in 47% of patients. The therapeutic potential of this regimen should be tested in patients with small-volume disease after debulking surgery.
To assess the prognostic value of tumor proliferative activity, 89 patients with operable non-small cell lung cancer were studied. Tumor samples were obtained during surgery and cell kinetics were analyzed by the in vitro thymidine labelling index (TLI). The overall median TLI (2.9) was used to identify two subsets of patients with high and low proliferating tumors. In univariate analysis survival was significantly longer in patients with lower TLI (P = 0.047) and with stage I-II (P = 0.003) and T1-T2 tumors (P = 0.043). In multivariate analysis, stage was the most important prognostic parameter (P = 0.004). The risk of death for patients with TLI higher than 2.9 was increased (hazard ratio = 2.01, CI = 0.96-4.27).
The tumor proliferative activity of 74 previously untreated epithelial ovarian carcinomas was evaluated by the thymidine labeling index (TLI) and the primer-dependent alpha DNA polymerase assay (PDP-LI). The median TLI and PDP-LI were 2% (0.1 to 28.0) and 10% (3.0 to 80.0), respectively. The TLI was significantly correlated to tumor grade, International Federation of Gynecology and Obstetrics (FIGO) stage, and residual disease. However, neither the TLI nor the PDP-LI was a predictor of survival. A higher response rate was observed in the case of rapidly proliferating tumors: an objective response (OR) was observed in 38.4% of the patients with a low TLI and in 53.6% of the patients with a high TLI. The advantage of an OR in favor of the high TLI group was significant for patients treated with regimens containing doxorubicin (high TLI, 72.2% OR; low TLI, 40% OR; P = 0.03).
The production of immunoreactive somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) by the established cell line derived from a human lung carcinoma CALU-6 has been evidenced in the serum-free medium in increasing concentrations as a function of the incubation time. Gel filtration in acid conditions of cell-conditioned medium collected after 72 h showed peaks of immunoreactive Sm-C/IGF-I in the elution volume corresponding to the molecular weight of the synthetic Sm-C/IGF-I, and in the high molecular weight region, where specific binding sites for Sm-C/IGF-I could be also demonstrated. These results indicate that this established cell line produces high amounts of immunoreactive Sm-C/IGF-I and of Sm-C/IGF-I carrier protein. The pooled fractions corresponding to the molecular weight of synthetic Sm-C/IGF-I showed a competitive binding curve parallel to the standard in the Sm-C/IGF-I RIA system, and a mitogenic activity on cells from the same line similar to the one observed using two different pure Sm-C/IGF-I preparations, obtained by chemical synthesis or by DNA recombinant technology. When a monoclonal antibody (sm-1.2) raised against Sm-C/IGF-I was added into the medium, the mitogenic effect observed by both synthetic and cell-derived Sm-C/IGF-I peptide was completely abolished; the monoclonal antibody also partially inhibited the effect of 10% fetal calf serum and the thymidine incorporation observed in serum-free medium without growth factors. In serum-free medium the monoclonal antibody produced a 45% reduction of cells in S phase by thymidine labeling index without modification of the growth fraction as determined by primer-dependent alpha-DNA polymerase labeling index. In conclusion it seems that Sm-C/IGF-I has a critical role in the autocrine stimulation of the replication of this cell line.
Thirty-nine patients with locally advanced breast cancer (T3b-4, N1-3 or inflammatory carcinoma) received 3 cycles of induction chemotherapy with estrogenic recruitment before surgery. The therapeutic regimen consisted of diethylstilbestrol (DES) orally on days 1-3, 5-Fluorouracil + Doxorubicin + Cyclophosphamide on day 4 q 21 days (DES-FAC). After surgery 6 additional cycles of chemotherapy (3 DES-FAC alternating with 3 DES-CMF with Methotrexate + F and C as in FAC) were administered. The objective response rate was 71.8% with 15.4% CR, and 56.4% PR; after surgery 36/39 (92.3%) patients were rendered disease-free. So far, 13 of 26 patients in stage IIIb have relapsed (9 of 13 with inflammatory carcinomas). Three-year survival and progression-free survival are 60% and 53.5%, respectively. Twenty-three of the 39 patients were subjected to serial tumor biopsies during the first DES-FAC regimen to allow for tumor-cell kinetic studies during DES and chemotherapy. A significant estrogenic recruitment occurred in 16 patients (69.6%), irrespective of estrogen-receptor status. At surgery, 3-4 weeks after induction chemotherapy, tumor proliferative activity was significantly depressed in comparison to basal values. These results indicate that breast cancer cells can be recruited in vivo with DES and that chemotherapy following estrogenic stimulation is effective and feasible with acceptable toxicity.
We have established and partially characterized a panel of monoclonal antibodies against alpha-DNA polymerase. One of the hybridomas, clone 5F, has been exploited for cell kinetic studies on three colon cancer cell lines, LOVO, SW 620, and SW 403, which are endowed with different growth patterns and differentiation status. By an immunoperoxidase method, we could demonstrate the specific intranuclear localization of alpha-DNA polymerase during the exponential phase of in vitro growth and contrast it with the diffuse distribution of the enzyme throughout the cytoplasm during the resting state. The percentage of intranuclear staining positive cells, evaluated at successive time points of in vitro growth, changed from 75 to 95% (assayed on Days 3 and 7) to 15 to 25% in confluent and resting populations assayed on Days 12 to 14. In agreement with the assumption that the enzyme moves from nucleus to cytoplasm after entering quiescence, alpha-DNA polymerase was still present in the cytoplasm or in the cytoplasmic perinuclear area of cells in resting phase cultures. Comparisons between traditional kinetic parameters (thymidine labeling index and primer-dependent alpha-DNA polymerase) and proliferative state determined by the monoclonal antibody supported the feasibility of this approach to define the proportion of actively proliferating elements in a tumor cell population. Moreover, parallel flow cytometric analysis performed on Days 5 and 14 of continuous culture showed fluctuations of alpha-DNA polymerase content in relation to exponential and steady-state phases, with a significant increase in the amount of alpha-DNA polymerase in actively proliferating populations and a progressive reduction of the enzyme as the cultures entered the resting stage.
Diethylstilbestrol (DES) can induce a recruitment into the proliferative pool of previously resting breast cancer cells in vivo. In order to verify if estrogenic recruitment could result in a larger tumor cell killing by chemotherapy, 117 patients with metastatic breast cancer were randomized to receive CEF (cyclophosphamide, 600 mg/m2; epidoxorubicin, 60 mg/m2; and 5-fluorouracil, 600 mg/m2 on day 1); DES-CEF (cyclophosphamide, 600 mg/m2 on day 1; DES, 1 mg orally on days 5, 6, and 7; and epidoxorubicin, 60 mg/m2, and 5-fluorouracil, 600 mg/m2, on day 8) every 21 days. No significant difference in objective response rates, survival, or progression-free survival was seen between the two regimens. Patients in the DES-CEF arm experienced a higher complete response (CR) rate (24.1% v 16.1%), which reached statistical significance in the case of soft-tissue metastasis (48% v 27.3%; P less than .05) and estrogen receptor-negative tumors (35.7% v 11.1%; P less than .025). Survival and progression-free survival of patients refractory to treatment were not worsened by estrogenic recruitment. In the subset of patients failing after adjuvant polychemotherapy, DES-CEF unexpectedly induced a significantly longer survival (greater than 802 days v 375 days; P = .029) and progression-free survival (239 days v 192 days; P = .041) than CEF. The DES-CEF regimen was more myelotoxic, and 43.3% of the DES-CEF cycles had to be delayed because of leukopenia in comparison with 11.8% of the CEF cycles (P less than .0001). In conclusion, chemotherapy with estrogenic recruitment was able to induce more CRs in certain subsets of patients and a significant prolongation in survival and progression-free survival of patients failing after adjuvant polychemotherapy. These results have been achieved despite a significantly lower dose intensity of chemotherapy.
Proliferating cells, independent of their position in the cell cycle, contain a primer-dependent alpha-DNA polymerase (PDP); we have, therefore, tested the feasibility of the PDP assay in breast, ovarian, and head and neck tumors. Experimental results demonstrate that the PDP labeling index (PDP-LI) is constantly superior to the thymidine labeling index (TLI), which indicates the percentage of S phase cells. The PDP-LI/TLI ratios observed were 5.0 in breast cancer, 3.6 in ovarian cancer, and 2.5 in head and neck tumors. Higher PDP-LI scores have been obtained in metastatic breast tumor samples (PDP-LI = 8.8) compared to nonmetastatic tumors (PDP-LI = 4.9).
Thirty-six patients with locally advanced breast cancer received diethylstilbestrol (1 mg/die) for 3 days followed by FAC (5 Fu 600 mg/m2, adriamycin 50 mg/m2, cytoxan 600 mg/m2) on day 4, q. 21 days. After three cycles, responsive patients were submitted to surgery. Tumor kinetic parameters were evaluated by TLI and PDP-LI in 22 patients on serial tumor biopsies at diagnosis (TO), after DES (T1), 24 hrs after the first FAC (T2) and at the time of radical surgery (T3). An estrogenic recruitment was evident by TLI in 9/22 tumors and by PDP-LI in 16/22 patients. Our results demonstrate that diethylstilbestrol can induce a kinetic recruitment of breast cancer cells independently from their ER content and that chemotherapy is able to stop cell proliferation.
We have evaluated the feasibility of a cytokinetically oriented regimen based on the induction of cell recruitment by diethylstilbestrol (DES) in locally advanced human breast cancer. Tumor proliferative activity was evaluated by the thymidine labeling index and the primer-dependent alpha-DNA polymerase labeling index, which gives an in vitro estimation of the growth fraction. Sixteen previously untreated patients received DES (1 mg daily for 3 days) followed by FAC [5-fluorouracil (600 mg/m2): Adriamycin (50 mg/m2): Cytoxan (600 mg/m2)] i.v. on day 4 every 21 days. Radical surgery was delayed to allow for three DES-FAC regimens in responsive patients. Proliferative activity on tumor biopsies was evaluated immediately before and after treatment with DES, 24 h after chemotherapy and, in nine patients, at the time of radical surgery. DES was able to induce a significant increase in thymidine labeling index in 8 of 16 patients, while the primer-dependent alpha-DNA polymerase labeling index was significantly increased in 13 of 16 tumors, independently of their estrogen receptor content. Subsequently administered chemotherapy induced an early decrease in tumor proliferation. In the nine patients submitted to surgery after three DES plus FAC courses, the average thymidine labeling index and primer-dependent alpha-DNA polymerase labeling index were 27.8 and 73% of the pretreatment values. Our preliminary results provide the rationale for the design of new therapeutic schemes in which antitumor drugs are given at the time of estrogen-induced tumor cell recruitment. Further extended studies are required to establish whether induction of tumor cell recruitment will actually translate into appreciable improvement of the clinical response to chemotherapy.
Kinetic recruitment of cancer cells can seldom be monitored in human solid tumors. Repeated tumor sampling in ascitic ovarian cancer has been exploited to study tumor cell kinetic recruitment following treatment with the alkylating agent iphosphamide (IFX). The treatment schedule of the study was designed to administer the antimetabolic agents MTX-5FU at the time of the recruitment peak. Kinetic studies by the labelling index (LI) assay could be performed during and after the IFX treatment in four out of eight patients because of sampling difficulties. Experimental results showed that the IFX effectiveness reduced the proliferating cells, followed by cell kinetic recruitment from the resting pool. The antimetabolic treatment in concomitance with the proliferative peak (day 12) has been reduced with respect to the original schedule due to the heavy leukopenia occurring to the patients. It is likely that the reduced drug dosage might have contributed to the poor clinical response. However, the recruited cells exhibited an increased in vitro chemosensitivity to adriamycin in comparison to tumor cells studied before the IFX treatment.
By co-cultivation procedures, infectious xenotropic type C viruses have been recovered from kidney cells of several strains of mice. They have host-range patterns which place them into separate subgroups. In cells cultivated from one NZB kidney, two biologically different xenotropic type C retroviruses were found. One, X-NZB/K-1, infects and replicates well in human and mink fibroblast cells but does not induce foci in mink S + L - cells with good efficiency. The other, X-NZB/K-2, infects and replicates well in mink but not human fibroblast cells, and induces foci readily in mink S + L - cells. Cross-infection studies indicate that these viruses, classified as xenotropic by host range and envelope properties, are genetically stable.
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The cytotoxic effect and antitumor activity induced by the novel organotin compound triethyltin(IV)lupinyisulfide hydrochloride, have been investigated. Different patterns of antiproliferative effects have been observed in a panel of human tumor cell lines in vitro. Toxicity studies in mice reported acute toxicity at the doses of 21 and 17.5 mg/kg which progressively disappeared at lower concentrations. On this basis, the doses of 3.5, 7 and 14 mg/kg were selected to assess the antitumor activity in vivo against the P388 leukemic cells xenografted in mice. This compound was able to induce a dose-dependent significant reduction of tumor volume, up to 46%, at the highest concentration (p = 0.0062) without important toxicity, as also confirmed by histological analysis of the main organ tissues. This preliminary study seems to hold interest for further investigations in different tumor models as well as for the evaluation of optimal drug route and schedule.
Indole-3-carbinol is a natural product which has been shown to reduce the incidence of spontaneous and carcinogen-induced mammary tumours in animals. Eighteen unsymmetrical methylene derivatives of indoles were prepared by reaction of Mannich bases of 7-hydroxycoumarins with substituted indoles in acetic or propionic anhydride. The synthesised molecules were tested in vitro against the MCF7 and MDA-MB-231 breast cancer cell lines by MTT and cell count assays. Results from 16 tested compounds showed that 60% of them exerted some effects against the MDA-MB-231 compared to about 30% towards the MCF7. Among all, the 3-(7'-acetoxy-4-methylcoumarin-8'-yl)methyl-2-methylindole resulted the most effective in both cell lines, compared to indole-3-carbinol. In conclusion, these preliminary results report that some of these compounds might be promising potential antiproliferative agents.
The effects induced by the antiandrogen Cyproterone Acetate (CPA) on the proliferation of EVSA-T human breast cancer cells endowed with androgen receptors were studied. Kinetic analyses were carried out by two autoradiographic techniques measuring the percentage of cells in S-phase and the growth fraction (GF). The exposure of the cultures to CPA for 24 h caused a marked inhibition on S-phase cells without significantly affecting the CF. The accumulation of cells in G1-phase, confirmed by cytometric analysis, was rescued to the S compartment by replacing the culture medium 24 h after CPA administration. Exposure of EVSA-T to Doxorubicin or Methotrexate after CPA and medium change at the time of maximal proliferative recruitment, led to an enhancement of cytotoxicity as demonstrated by colony survival assay.
A hybridoma cell line (5F) secreting monoclonal antibodies directed to alpha DNA polymerase has been developed. Kinetic studies on peripheral blood lymphocytes stimulated with mitogen and human colon cancer cell lines established in vitro were made by the two autoradiographic techniques of Thymidine Labelling Index and Primer-dependent alpha DNA polymerase Labelling Index and the immunoperoxidase assay (PAP) with monoclonal antibody to alpha DNA polymerase. We demonstrated the exclusively intranuclear presence of alpha DNA polymerase in lymphocytes induced to proliferate and actively growing colon cancer cells in contrast with the cytoplasmic distribution of the enzyme in resting stage populations. The feasibility of using monoclonal antibodies to alpha DNA polymerase to determine cell growth fraction was evaluated.