Sequential dose-dense Doxorubicin and Ifosfamide in advanced soft-tissue sarcoma patients in an out-patient-basis schedule.

Aims. This phase II study explored activity/safety of front-line dose-dense chemotherapy in high-grade STS (soft tissue sarcoma) patients and tested ezrin as prognostic factor. Patients and Methods. The protocol consisted of three cycles of doxorubicin (DOXO) 30 mg/m(2) on days 1-3 every 2 weeks, followed by three cycles of ifosfamide (IFO) 2.5 g/m(2) two hours a day on days 1-5 every 3 weeks, with GCSF support. Ezrin was assessed immunohistochemically. Results. Twenty patients, 13 metastatic and 7 locally advanced, were enrolled. Median age was 39 years (25-60). Median dose intensities were 42 mg/m(2)/week and 3.6 g/m(2)/week for DOXO and IFO, respectively. Grade 3/4 toxicities occurred in 18 patients. Response rate was 15% (3 of 20) by RECIST. Patients younger than 45 years with locally advanced disease and synovial histology presented longer survival. A trend towards longer survival was observed among ezrin-positive patients. Conclusions. This dose-dense schedule should not be routinely used due to its high frequency of toxic events; however, a sequential strategy with DOXO and IFO may benefit selected patients and should be further explored with lower doses. The role of ezrin as a prognostic marker should be confirmed in a larger group of patients.

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1)

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of ß1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5 percent dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha2 (63.8 ± 11.3 percent positive cells), alpha3 (93.3 ± 7.0 percent), alpha5 (50.4 ± 12.0 percent) and alpha6 (34.1 ± 4.9 percent) integrins but not alpha1, alpha4, alphav or ß4. Cells adhered well to laminin-1 (73.4 ± 6.0 percent) and fibronectin (40.0 ± 2.0 percent) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha2, alpha3 and alpha6 mediated laminin-1 adhesion, but neither alpha3 nor alpha5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 ± 2.4 percent vs DMSO: 70.7 ± 2.5 percent) while simultaneously reducing alpha5 (24.2 ± 19 percent) and alpha6 (14.3 ± 10.8 percent) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha3 and alpha5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 ± 2 cells vs DMSO: 64 ± 6 cells), was blocked by an antibody against alpha6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells

Simultaneous changes in the function and expression of beta 1 integrins during the growth arrest of poorly differentiated colorectal cells (LISP-1).

Cells usually lose adhesion and increase proliferation and migration during malignant transformation. Here, we studied how proliferation can affect the other two characteristics, which ultimately lead to invasion and metastasis. We determined the expression of beta 1 integrins, as well as adhesion and migration towards laminin-1, fibronectin, collagens type I and type IV presented by LISP-1 colorectal cancer cells exposed to 2.5% dimethyl sulfoxide (DMSO), an agent capable of decreasing proliferation in this poorly differentiated colorectal cell line. Untreated cells (control), as shown by flow cytometry and monoclonal antibodies, expressed alpha 2 (63.8 11.3% positive cells), alpha 3 (93.3 7.0%), alpha 5 (50.4 12.0%) and alpha 6 (34.1 4.9%) integrins but not alpha1, alpha 4, alpha v or 4. Cells adhered well to laminin-1 (73.4 6.0%) and fibronectin (40.0 2.0%) substrates but very little to collagens. By using blocking monoclonal antibodies, we showed that alpha 2, alpha 3 and alpha 6 mediated laminin-1 adhesion, but neither alpha 3 nor alpha 5 contributed to fibronectin adherence. DMSO arrested cells at G0/G1 (control: 55.0 2.4% vs DMSO: 70.7 2.5%) while simultaneously reducing alpha 5 (24.2 19%) and alpha 6 (14.3 10.8%) expression as well as c-myc mRNA (7-fold), the latter shown by Northern blotting. Although the adhesion rate did not change after exposure to DMSO, alpha 3 and alpha 5 played a major role in laminin-1 and fibronectin adhesion, respectively. Migration towards laminin-1, which was clearly increased upon exposure to DMSO (control: 6 2 cells vs DMSO: 64 6 cells), was blocked by an antibody against alpha 6. We conclude that the effects of DMSO on LISP-1 proliferation were accompanied by concurrent changes in the expression and function of integrins, consequently modulating adhesion/migration, and revealing a complex interplay between function/expression and the proliferative state of cells.

Transforming growth factor beta1, urokinase-type plasminogen activator and plasminogen activator inhibitor-1 mRNA expression in head and neck squamous carcinoma and normal adjacent mucosa.

BACKGROUND: A balance between urokinase-type plasminogen activator (uPA) and its main inhibitor type-1 (PAI-1) appears to be important for cancer invasive behavior. Since uPA/PAI-1 system seems to be regulated by transforming growth factor beta1 (TGFbeta1) in different cell types, our aim was to investigate the relationship between the expression of the three genes and lymph node status in head and neck squamous cell carcinomas (HNSCC) at specific sites. MATERIALS AND METHODS: uPA, PAI-1, and TGFbeta1 mRNAs were determined by Northern analysis in tumor, and paired normal mucosa samples were obtained from 91 operable HNSCC patients. RESULTS: In oral cavity, excluding tongue, TGFbeta1, PAI-1, and uPA mRNAs values were consistently lower in the normal tissues than in tumors. In larynx tumors, TGFbeta1 expression was increased, but no statistically significant differences were found for uPA or PAI-1 mRNAs as compared with normal tissues. Tongue tumors overexpressed only uPA mRNA, and uPA levels showed significant parallel variations with TGFbeta1 and PAI-1 mRNAs mainly in pN+ tumors. In oral cavity tumors, an inverse correlation between TGFbeta1 and uPA was observed in pN0 subgroup, elevated uPA mRNA was counterbalanced by high PAI-1 mRNA TGFbeta1, and PAI-1 were not coordinately expressed. Correlations between the three markers were not found in larynx. Hypopharynx tumors, all staged as pN+, expressed the lowest TGFbeta1 mRNA mean values. CONCLUSIONS: Combined information about TGFbeta1, uPA, and PAI-1 mRNAs may add some clues to the understanding of the pathophysiological role of uPA system in head and neck squamous cell carcinoma.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest.

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Eosinophil accumulation in rat uterus following estradiol administration is modulated by laminin and its integrin receptors.

Eosinophils accumulate into the uterus of ovariectomized rats, after treatment with estradiol (E2). We have investigated whether this feature is related to interactions of eosinophils with uterine extracellular matrix proteins: laminin (LM) and fibronectin (FN). Eosinophils isolated from the peritoneal cavity of ovariectomized rats displayed estrogen receptors measured at both binding activity and mRNA levels. An increased number of laminin binding sites, calculated by Scatchard analysis using iodinated LM was determined in E2-treated eosinophils (70,100 +/- 28,000 sites/cell vs 21,000 +/- 5,000 sites/cell in controls). Eo binding to 125I-LM- was inhibited by the E8-LM fragment. Estradiol up-regulated the expression in eosinophils of alpha 6 and beta 2 integrin subunits evaluated by flow-cytometry as well as by alpha 6 mRNA expression. After E2 treatment, eosinophils showed higher adhesiveness to LM-coated dishes (10 +/- 2 vs 56 +/- 3%) which was inhibited by monoclonal antibodies against alpha 6, beta 1 and beta 2 integrins and by the steroid antagonist tamoxifen. These monoclonal antibodies also blocked the attachment of stimulated eosinophils to uterine cryostat sections obtained from spayed rats previously treated with estradiol. We did not detect any apparent influence of E2 on basal eosinophil adherence or binding to FN although alpha 4 and alpha 5 integrin subunits were expressed in eosinophils. Expression of laminin and merosin in the uterus was determined immunohistochemically. Our results suggest that integrin-laminin interactions may contribute to the preferential eosinophil recruitment in vivo.

Expression of vitamin D receptor (VDR) in HL-60 cells is differentially regulated during the process of differentiation induced by phorbol ester, retinoic acid or interferon-gamma.

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.

Integrin receptors and TGF-beta expression in chronic myeloid leukemia cells.

To understand the relationship between transforming growth factor beta-1 (TGF-beta 1) and the integrin profile presented by chronic myeloid leukemia cells, we have studied, using Northern analysis, the expression of TGF-beta 1 messenger RNA (TGF-beta mRNA) in myeloid cell lines and in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In addition we determined the positivity for alpha 4 and alpha 5 integrin molecules in those cells using specific monoclonal antibodies and flow cytometry. CML patients (N = 3) presented mean values of alpha 4 and alpha 5 higher (alpha 4: 60 +/- 20%; alpha 5: 70 +/- 41%) than AML (N = 10) blast cells (alpha 4: 25 +/- 23%; alpha 5: 18 +/- 16%). Northern analysis revealed an almost four-fold higher expression of TGF-beta mRNA in K562 (derived from a patient with chronic myeloid leukemia) compared to the myeloblastic cell line HL60. The highest TGF-beta mRNA levels were seen in the U937 lineage. CML leukemic cells (N = 3) showed high TGF-beta mRNA levels comparable to the levels expressed by K562 which was paralleled by high beta 1 integrin mRNA. AML blast cells presented a variable degree of expression of TGF-beta mRNA when compared to HL60. One patient with acute megakaryoblastic leukemia (FAB subtype M7), usually associated with myelofibrosis, presented the highest TGF-beta mRNA levels. We conclude that studying TGF-beta 1 and its mechanisms of action will help in understanding fibrosis in leukemic patients, and perhaps to design treatments for such conditions.

Integrin receptors and TGF-Beta expression in chronic myeloid leukemia cells

To understand relationiship between transforming growth factor beta-1 (TGF-ß1) and the integrin profile presented by chronic myeloid leukemia cells, we have studied, using Northen analysis, the expression of TGF-ß1 messenger RNA (TGF-ß mRNA) in myeloid cell lines and in patient with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In addition we determined the positivity for alfa4 and alfa5 integrin moleculas in those cell using specific monoclonal antibodies and flow cytometry. CML patients (N=3) presented mean values of alfa4 higher (alfa4: 60 ñ 20 per cent); alfa5: 70 ñ 41 per cent) than AML (N=10) blast cells (alfa4: 25 ñ 23 per cent); alfa5: 18 ñ 16 per cent). Northern analysis revealed an almost four-fold higher expression of TGF-ß mRNA in K562 (derived from a patient with chronic myeloid leukemia) compared to the myeloblastic cell line HL60. The highest TGF-ß mRNA levels were seen in the U937 lineage. CML leukemic cells (N=3) showed high TGF-ß mRNA levels comparable to the levels expressed by K562 which was paralleled by high ß1 integrin mRNA. AML blast cells presented a variable degree of expression of TGF-ß mRNA when compared to HL60. One patient with acute megakaryoblastic leukemia (FAB subtype M7), usually associated with myelofibrosis, presented the highest TGF-ß mRNA levels. We conclude that studing TGF-ß1 and its mechanisms of action will help in understanding fibrosis in leukemic patients, and perhaps to design treatments for such conditions

Differential expression of fibronectin receptors during myeloid differentiation and in marrow- and peripheral blood-derived leukemic cells.

A 125I-labeled 120-kDa fibronectin fragment (FN120) containing the RGD binding site was employed to assess FN120 receptor levels in control and dimethylsulfoxide (DMSO)-differentiated HL60 cells, as well as in leukemic peripheral and bone marrow blast cells from acute lymphoid (ALL) and myeloid (AML) patients. Fibronectin CS1 fragment receptor alpha 4 (VLA4-alpha) and RGD-dependent alpha 5 integrin subunits (VLA5-alpha) were characterized by specific monoclonal antibodies (MoAb). HL60 cells, induced along the granulocytic pathway with DMSO, displayed low FN120 binding level densities (36,070 +/- 5142 sites/cell (s/c) vs. 19,780 +/- 4564 s/c, P < 0.005), respectively, for untreated and treated cells) together with decreased VLA5-alpha expression. Granulocytes displayed low levels of FN120 receptors (3167 +/- 1165 s/c) with weak VLA5-alpha expression and absence of VLA4-alpha. Normal lymphocytes displayed 17,670 +/- 8,705 s/c FN120 receptors and VLA4-alpha and VLA5-alpha. The mean FN120 binding levels and mean VLA5-alpha expression were lower in peripheral blast cells, both in ALL and AML, than in the bone marrow leukemic cells. VLA4-alpha remained the same irrespective of cell localization. FN120 binding sites and differential expression of VLA4-alpha and VLA5-alpha integrin molecules on hemopoietic cells could be related to lineage characteristics or cell type distribution within hemopoietic tissue.

Vitamin D3 binding activity during leukemic cell differentiation.

In this paper we report that differentiation of the human promyelocytic leukemia cell line, HL60, along the myelocytic pathway, induced by retinoic acid (RA), or monocytic pathway, induced by phorbol-myristate acetate (PMA) and gamma interferon (IFN), was accompanied by a significant decline in 1,25-dihydroxycholecalciferol (1,25(OH)2D3) binding (control: 30.3 +/- 3.0 fM/10(6) cells; RA treated: 6.8 + 2.5 fM/10(6) cells; PMA treated: 12.3 +/- 6.7 fM/10(6) cells and IFN treated: 16.0 +/- 5.0 fM/10(6) cells). When differentiation and proliferation were uncoupled, by incubation with IFN or by inhibition of proliferation by cell density saturation, 1,25(OH)2D3 binding was better related to differentiation than to proliferation. Additionally we have compared 1,25(OH)2D3 binding levels in blasts from acute lymphocytic leukemia (ALL) patients (25.4 +/- 18.1 fM/10(6) cells) and normal, mature lymphocytes (10.6 +/- 2.1 fM/10(6) cells). Receptor binding was significantly higher (p < 0.05) in the immature blasts. Our data suggest that 1,25(OH)2D3 receptor levels could be considered a marker of functional immaturity, in these cells.

Increased expression of laminin receptors during myeloid differentiation.

Variation of laminin receptor levels (LNR) during myeloid-cell differentiation and in acute leukemia were investigated by 125I-laminin-binding determination during HL60 cell differentiation and in cells of patients with different types of leukemia, characterized according to the FAB classification. LNR levels in HL60 cells increased during differentiation, being significantly higher in cells exposed to phorbol myristate acetate (PMA) and ethanol (55,391 +/- 27,845 and 29,314 +/- 6,435 sites/cell respectively) as compared with HL60 controls (8,549 +/- 4,000 sites/cell). The control cells do not adhere to laminin-coated surfaces, but differentiation with PMA results in their rapid adherence on this substratum. Short treatment with PMA does not increase the number of adherent cells or the receptor expression. Granulocytes also presented equally high LNR concentration (29,739 +/- 13,516 sites/cell). The lymphoid cells (lymphocyte, acute lymphoid leukemia and chronic lymphocytic leukemia) shared low LNR numbers (less than 6,500 sites/cell). Myeloid cells displayed a wide range of LN receptors with higher levels being associated with the more differentiated FAB subgroups. 125I-laminin binding to lymphoid or myeloid leukemic cells was mainly inhibited by P1 fragments, whereas granulocytes and differentiated HL60 cells displayed a dual binding pattern for laminin fragments P1 and E8. These results were confirmed by assays using 125I-labelled P1 and E8 fragments. We conclude that magnitude of LNR levels and variation in expression of P1 and E8 receptors appear to be linked to lineage and maturation status in hematopoietic cells.

Glucocorticoid receptors in acute leukemia.

We evaluated the applicability of glucocorticoid receptor (GR) determinations to predict clinical responsiveness to polychemotherapy in acute leukemias by measuring GR in leukemic cells from 20 patients as well as in lymphocytes from 20 normal volunteers. The whole-cell binding assay with [3H]-dexamethasone was used. The GR level (mean +/- SD) was 4583 +/- 1384 sites/cell (range: 2050-8140) for normal lymphocytes. A significant amount of GR (5300 to 17,000 sites/cell) was detected in the blasts from 9/12 patients with acute lymphoblastic leukemia (ALL). The concentration of GR sites in ALL cells greatly exceeded that found in normal mononuclear cells (P less than 0.01). The absence of GR in ALL patients correlated with poor response to polychemotherapy including glucocorticoids. High receptor levels were associated with complete remission (P less than 0.005). The GR concentration (7288 +/- 2345 sites/cell) found in acute non-lymphoblastic leukemias (ANLL) was in the same range as that found in ALL cases. All ANLL patients had a substantial number of GR, significantly higher than the sites/cell found in normal lymphocytes (P less than 0.05). No correlation between clinical responsiveness and receptor level was demonstrable for ANLL patients.