Interleukin-1 beta, transforming growth factor beta 1, prostaglandin E2, and fibronectin levels in the conditioned mediums of bone marrow fibroblast cultures from lung and breast cancer patients.

We analyzed the ability of the bone marrow (BM) stromal cells to achieve confluence and their proliferative capacity in BM primary cultures from 30 untreated lung cancer patients (LCP), 27 breast cancer patients (BCP), and 30 normal controls (NC) when these confluent cells were induced to proliferate following four continuous subcultures. Moreover, we evaluated the production of interleukin-1 beta (IL-1beta), transforming growth factor beta 1 (TGF-beta1), fibronectin, and prostaglandin E2 (PGE2) by pure fibroblasts (fourth passage). A fibroblast colony-forming units (CFU-F) assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta, TGF-beta1, and fibronectin in conditioned mediums (CM) of fibroblast cultures were measured by enzyme-linked immunosorbent assay (ELISA) kit and PGE(2) by radioimmunoassay (RIA) kit. Confluence was achieved in the 60% of LCP and 78% of BCP primary cultures compared with 100% of NC, and only fibroblasts from seven LCP and six BCP cultures had the capacity to proliferate following four subcultures. Levels of IL-1beta were below 10 pg/ml in both patient groups, while NC had a mean value of 5882.57+/-221.61 pg/ml. Levels of TGF-beta1 in BCP were lower than NC values ( P<0.05). LCP and BCP had significantly decreased levels of fibronectin when compared to NC values ( P<0.05 and P<0.01, respectively). Levels of PGE2 in LCP were higher compared to NC ( P<0.01). In conclusion, BM fibroblasts from LCP and BCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta, TGF-beta1, fibronectin, and PGE2 production.

Interferon-alpha 2b modulation of doxorubicin sensitivity in a multidrug resistant cell line.

We evaluated the effect of interferon-alpha2b as a chemosensitiser on HCT-15 cell line in treatment with doxorubicin. Chemosensitivity was determined by [3H]-thymidine incorporation and tetrazolium assays. The levels of expression of P-glycoprotein, Bcl-2 oncoprotein and HLA-ABC complex, and cell cycle/apoptosis analysis were determined by flow cytometry. Dox 50 ng/ml - IFN alpha 2b 500 IU/ml treatment inhibited cell proliferation (47.2 +/- 1.4%, p < 0.0001; MTT assay: 40.6 +/- 1.2%, p < 0.0001) and augmented the expression of P-170, Bcl-2 and HLA-ABC, while it didn't exert apoptosis, producing a slight G2/M arrest. A concentration of IFN-alpha2b, that by itself is not cytotoxic, can potentiate the efficacy of the anticancer drug. This effect is not due to a down-modulation of P-170. The absence of apoptosis and augmented levels of Bcl-2 expression suggests that this could be one of the mechanisms of drug resistance exerted by these cells.

Bone marrow fibroblasts in patients with advanced lung cancer

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1ß and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46 percent) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100 percent). Levels of spontaneously released IL-1ß were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 + or - 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 + or - 23.6 pg/ml) compared to NC (18.5 + or - 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1ß and PGE2 production

Bone marrow fibroblasts in patients with advanced lung cancer.

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1beta (IL-1beta) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46%) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100%). Levels of spontaneously released IL-1beta were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 +/- 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 +/- 23.6 pg/ml) compared to NC (18.5 +/- 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta and PGE2 production.

Tumor necrosis factor-alfa production by monocytes from lung and colorectal cancer patients.

Tumour necrosis factor-alpha (TNF-alpha) is a monocyte (MO)-derived cytokine that plays an essential role in the immunological system. In the present study our aim was to evaluate the levels of TNF-alpha secreted by MO from cancer patients. Blood MO were obtained from 10 lung cancer patients (LCP), 10 colorectal cancer patients (CCP) and 10 healthy donors (HD). TNF-alpha levels in MO culture supernatants spontaneously (sp) secreted or after stimulation with LPS treatment were evaluated using a commercial ELISA kit (sensibility: 10-1000 pg/ml). Mean values, expressed as pg/ml were: LCP: sp= 452.6+/-107.2, LPS= 589.5+/-126.7); CCP: sp= 84.1+/-25.0, LPS= 437.3+/-93.2; HD: sp= 74.2+/-21.5, LPS= 573.5+/-87.1. We concluded that MO from LCP secrete high levels of TNF-alpha spontaneously (p< 0.003 versus HD) and it was also observed an absence of response to LPS treatment in the 33% of the cases in these patients.

In vitro secretion of cytokines and prostaglandin-E2 by monocytes from lung cancer patients.

Monocytes (MO) from cancer patients present functional abnormalities, such as an altered secretion of soluble factors. In the present study, our aim was to evaluate the levels of interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and prostaglandin-E2 (PGE2) secreted in vitro by MO from lung cancer patients (LCP), spontaneously or after stimulation with lipopolysaccharide (LPS). Results showed that cytokine secretion was higher for MO from LCP than for MO from healthy controls, while in the 25% of the patients analysed, an absence of response to LPS treatment was found.

Regulation of HLA-DR antigen in monocytes from colorectal cancer patients by in vitro treatment with human recombinant interferon-gamma.

During the immune response, a great number of cytokines that modulate the function of mononuclear phagocytes are produced. Since interferons are one of the most important cytokines, the aim of this study was to evaluate the expression of HLA-DR antigen after an 18-h culture with human recombinant interferon-gamma (hrIFN-gamma) on freshly isolated peripheral blood monocytes from 16 colorectal cancer patients and 16 healthy donors, using an indirect immunofluorescence method. The results obtained showed that there was a decreased percentage of HLA-DR+ monocytes in the colorectal cancer patents (51 +/- 3%, p <0.01) compared with the healthy donors (77 +/- 2%). Treatment for 18 h with hrIFN-gamma increased the percentages of monocytes expressing HLA-DR: 71 +/- 3% for the cancer patients and 84 +/- 2% for the healthy donors (p <0.01 and <0.001, respectively). Our results demonstrate that after in vitro treatment with hrIFN-gamma, functionally altered monocytes from colorectal cancer patients can reach major histocompatibility complex II antigen expression values similar to those of healthy donors, thus improving the host's cellular immune response against the tumor.

Cisplatin containing chemotherapy influences HLA-DR expression on monocytes from cancer patients.

The effect of cisplatin-containing chemotherapy regimen was evaluated on the expression of HLA-DR antigen in peripheral blood monocytes from patients with lung (LCP) and colorectal (CCP) cancer. Chemotherapeutic schedules employed in patients were etoposide and cisplatin for LCP and 5-fluorouracil and cisplatin for CCP. The results obtained showed a diminished percentage of monocytes expressing HLA-DR antigen in LCP (52.4 +/- 2.6, p < 0.004) and CCP (50.1 +/- 2.1, p < 0.001) respectively versus healthy donors (71.0 +/- 1.1%), and that their values increased during chemotherapy, raising them up to control level after the second cycle of treatment, independently of the course of the cancer growth. We conclude that both modalities of treatment allowed an increase of monocytes expressing HLA-DR antigen, suggesting that this effect may be due to cisplatin action.

Bone marrow fibroblastic progenitors in patients with advanced breast cancer.

Bone marrow fibroblast colony-forming cells (CFU-F) were studied in fifteen consecutive untreated breast cancer patients (BCP) with clinical stages III and IV, and in sixteen normal controls (NC). A decreased number of CFU-F was observed in BCP compared to NC (p < 0.004). Confluence of the adherent cell layer was observed in all normal bone marrow mononuclear cells (MC) cultures, while a lower proportion of cultures from BCP (11/15) showed confluent adherent cell layers. When MC cultures of BCP were treated with indomethacin (Indo, 10(-6)M) 50% of them increased the number of CFU-F compared to the value obtained without treatment. In addition, a significant increase in the release of PGE2 in BCP cultures was observed before Indo treatment. Moreover, after MC were fractionated into adherent and non-adherent progenitors, the CFU-F decreased in both types of fractions of BCP compared to NC value (p < 0.02 and < 0.05, respectively). The number of light density MC per 10 ml of bone marrow aspirate and the number of trypsin-sensitive adherent progenitors were lower than NC in BCP (p < 0.02 and 0.013, respectively). Total MC and fibroblasts (fourth passage) were cultivated to evaluate the production of interleukin-1 beta (IL-1 beta) by ELISA methodology. Results indicated no difference of IL 1 beta spontaneous release when total MC cultures of both groups were compared. However, the levels of this cytokine were lower (< 10 pg/ml) in fibroblast culture supernatants of BCP compared to NC (1,217 +/- 74 pg/ml). Fibroblast cultures from BCP showed low or no release of IL-1 beta after muramyl-dipeptide (MDP. 1 microgram/ml) stimulation. In conclusion, the defective proliferative and confluence capacity of BCP fibroblastic progenitors may be related to the decrease in the production of IL-1 beta by these precursors.

Fibroblastic colony-forming units and levels of tumor necrosis factor and prostaglandin E2 in bone marrow cultures from patients with advanced lung carcinoma.

BACKGROUND: Although alterations of the bone marrow (BM) fibroblast colony-forming cells are involved in the development of diverse hematologic disorders, these progenitors still have not been well characterized in patients with solid tumors. METHODS: The incidence of fibroblast colony-forming units (CFU-F) was evaluated in the cultures of unseparated and fractionated light density BM mononuclear cells (MC) from 25 consecutive untreated lung carcinoma patients (LCP) and 16 normal controls (NC). Unseparated MC also were cultured in the presence of indomethacin (10[-6] M). Finally, the authors evaluated the spontaneous production of prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) in culture conditioned mediums of unseparated MC by radioimmunoassay and enzyme-linked immunoadsorbent assay methodology, respectively. RESULTS: A decreased number of CFU-F was observed in unseparated and fractionated (adherent and nonadherent) light density MC cultures from LCP compared with NC. When unseparated MC of LCP were treated with indomethacin, a slightly increase in the number of CFU-F was found. Adherent MC (stromal cells) achieved confluence only in 44% of LCP primary cultures compared with 100% of NC. Overproduction of PGE2 and TNF-alpha was found in the conditioned mediums of LCP compared with the mean values obtained in NC (P < 0.05 and P < 0.02, respectively). CONCLUSIONS: The lack of confluence and suppression of CFU-F in BM of LCP may be related to the increase production of PGE2 and TNF-alpha. Future investigation will allow the determination of how these modifications influence tumor cell growth and will prove if more alterations of the hematopoietic microenvironment imply a worse prognosis.