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Purpose: The complete molecular basis of the organ-specificity of metastasis is elusive. This study aimed to provide an independent characterization of the transcriptional landscape of breast cancer metastases with the specific objective to identify liver metastasis-selective genes of prognostic importance following primary tumor diagnosis., Experimental Design: A cohort of 304 women with advanced breast cancer was studied. Associations between the site of recurrence and clinicopathologic features were investigated. Fine-needle aspirates of metastases (n = 91) were subjected to whole-genome transcriptional profiling. Liver metastasis-selective genes were identified by significance analysis of microarray (SAM) analyses and independently validated in external datasets. Finally, the prognostic relevance of the liver metastasis-selective genes in primary breast cancer was tested., Results: Liver relapse was associated with estrogen receptor (ER) expression (P = 0.002), luminal B subtype (P = 0.01), and was prognostic for an inferior postrelapse survival (P = 0.01). The major variation in the transcriptional landscape of metastases was also associated with ER expression and molecular subtype. However, liver metastases displayed unique transcriptional fingerprints, characterized by downregulation of extracellular matrix (i.e., stromal) genes. Importantly, we identified a 17-gene liver metastasis-selective signature, which was significantly and independently prognostic for shorter relapse-free (P < 0.001) and overall (P = 0.001) survival in ER-positive tumors. Remarkably, this signature remained independently prognostic for shorter relapse-free survival (P = 0.001) among luminal A tumors., Conclusions: Extracellular matrix (stromal) genes can be used to partition breast cancer by site of relapse and may be used to further refine prognostication in ER positive primary breast cancer., (©2015 American Association for Cancer Research.)

The primary objective was to estimate serum thymidine kinase 1 (TK1) activity, reflecting total body cell proliferation rate including cancer cell proliferation, in women with loco regional inoperable or metastatic breast cancer participating in a prospective and randomized study. Secondary objectives were to analyze TK1 in relation to progression-free survival (PFS), overall survival (OS), therapy response and other tumour characteristics, including CA 15-3, widely used as a standard serum marker for disease progression. TK1 and CA 15-3 were analysed in 198 serum samples collected prospectively from women included in the randomized TEX trial between December 2002 and June 2007. TK1 activity was determined by the ELISA based DiviTum™ assay, and CA 15-3 analyses was generated with the electrochemiluminescence immunoassay Cobas Elecsys CA 15-3 II. High pre-treatment TK1 activity predicted shorter PFS (10 vs. 15 months p = 0.02) and OS (21 vs. 38 months, p < 0.0001), respectively. After adjustment for age, metastatic site and study treatment TK1 showed a trend as predictor of PFS (p = 0.059) and was an independent prognostic factor for OS, (HR 1.81, 95 % confidence interval (CI) 1.26-2.61, p = 0.001). There was a trend of shortened OS for women with high CA 15-3 (p = 0.054) in univariate analysis, but not after adjustment for the above mentioned covariates. Both TK1 (p = 0.0011) and CA 15-3 (p = 0.0004) predicted response to treatment. There were statistically different distributions of TK1 and CA 15-3 in relation to the site of metastases. TK1 activity measured by DiviTum™ predicted therapy response, PFS and OS in loco regional inoperable or disseminated breast cancer. These results suggest that this factor is a useful serum marker. In the present material, a prognostic value of CA 15-3 could not be proven.

Most patients with advanced cancer experience severe pain and are often treated with opiates. Cancer patients are especially susceptible to opportunistic infections due to treatment with immunosuppressive and cytostatic drugs. Since opiates have been demonstrated to have immunomodulatory effects, it is of clinical importance to evaluate potential differences between commonly used opiates with regard to their effect on the immune system. The aim of this study was to evaluate the effect of morphine, tramadol, fentanyl and ketobemidone on the functioning of the immune system with special reference to TNF and IL-8 release. Method. U-937 cells were preincubated with different concentrations of opioids followed by stimulation with LPS 100 μg/ml for three hours. The effect of opioids on the levels of cytokine mRNA was studied using RT-PCR. Erk and Akt phosphorylation was also measured by Western blot. Results. All opioids with the exception of fentanyl were capable of inhibiting TNF release from U-937 cells. Morphine had no effect on IL-8 release but the effect of other opiates was almost the same as the effect on TNF. All opioids with the exception of fentanyl were capable of inhibiting production of mRNA for TNF and IL-8. The observed effects of opiates were not always reversible by naloxone, suggesting that the effects might be mediated by other receptors or through a non-receptor mediated direct effect. Although preliminary evidence suggests the involvement of Erk and Akt pathways, further studies are needed to unravel the intracellular pathways involved in mediating the effects of opiates. Our data suggests that the order of potency with regard to inhibition of cytokine release is as follows: tramadol > ketobemidone > morphine > fentanyl. Conclusion. Further studies are needed to understand the clinical implications of the observed immunosuppressive effects of tramadol and ketobemidone and to improve opioid treatment strategies in patients with cancer.

The aim of this was to determine whether the change of size observed at the first response evaluation after initiation of first-line combination chemotherapy correlates with overall survival (OS) in patients with metastatic breast cancer (MBC). The change in size of tumors derived from measurements according to Response Evaluation Criteria In Solid Tumors (RECIST) at the first evaluation on computed tomography (CT) was obtained from a multicenter, randomized phase III trial ("TEX trial," n = 287) comparing treatment with a combination of epirubicin and paclitaxel alone or with capecitabine (TEX). Cox regression and Kaplan-Meier analyses were performed to evaluate the correlations between the first change in tumor size, response according to RECIST and OS. Data from CT evaluations of 233 patients were available. Appearance of new lesions or progression of non-target lesions (new/non-target) indicated short OS by univariable regression analysis (HR 3.76, 95 % CI 1.90-7.42, p < 0.001). A decrease by >30 % at this early time point was prognostic favorable (HR 0.69, 95 % CI 0.49-0.98, p = 0.04) and not significantly less than the best overall response according to RECIST. After adjustment for previous adjuvant treatment and the treatment given within the frame of the randomized trial, OS was still significantly shorter in patients with new/non-target lesions after a median 8 weeks of treatment (HR 4.41, 95 % CI 2.74-7.11, p < 0.001). Disease progression at the first evaluation correlates with OS in patients with MBC treated with first-line combination chemotherapy. The main reason for early disease progression was the appearance of new lesions or progression of non-target lesions. These patients had poor OS even though more lines of treatment were available. Thus, these factors should be focused on in the response evaluations besides tumor size changes.

Anthracyclines and taxanes are active cytotoxic drugs in the treatment of early metastatic breast cancer. It is yet unclear whether addition of capecitabine to the combination of these drugs improves the treatment outcome. Patients with advanced breast cancer were randomized to first-line chemotherapy with a combination of epirubicin (Farmorubicin(®)) and paclitaxel (Taxol(®)) alone (ET) or in combination with capecitabine (Xeloda(®), TEX). Starting doses for ET were epirubicin 75 mg/m(2) plus paclitaxel 175 mg/m(2), and for TEX epirubicin 75 mg/m(2), paclitaxel 155 mg/m(2), and capecitabine 825 mg/m(2) BID for 14 days. Subsequently, doses were tailored related to side effects. Primary endpoint was progression-free survival (PFS); secondary endpoints were overall survival (OS), time to treatment failure (TTF), objective response (OR), safety and quality of life (QoL). 287 patients were randomized, 143 to ET and 144 to TEX. Median PFS was 10.8 months for patients treated with ET, and 12.4 months for those treated with TEX (HR 0.84, 95% CI 0.65-1.07, P = 0.16); median OS was 26.0 months for women in the ET versus 29.7 months in the TEX arm (HR 0.84, 95% CI 0.63-1.11, P = 0.22). OR was achieved in 44.8% (ET) and 54.2% (TEX), respectively (χ(2) 3.66, P = 0.16). TTF was significantly longer for patients treated with TEX, 6.0 months, versus 5.2 months following ET (HR 0.73, 95% CI 0.58-0.93, P = 0.009). Severe hematological side effects related to epirubicin and paclitaxel were evenly distributed between the treatment arms, mucositis, diarrhea, and Hand-Foot syndrome were significantly more frequent in the TEX arm. Toxicity-adjusted treatment with ET and TEX showed similar efficacy in terms of PFS, OS, and OR. In this trial with limited power, the addition of capecitabine to epirubicin and paclitaxel as first-line treatment did not translate into clinically relevant improvement of the outcome.

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC(50)) was approximately 0.8 μM and by 5 μM both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 μM canertinib accumulated the cells in the G(1)-phase of the cell cycle within 24h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 μM canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses ≤ 5μM canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recent findings have indicated that tyrosine kinase inhibitors (TKIs) targeting the ERBB receptor family display anti-leukaemic effects, despite the lack of receptor expression on human leukaemic cells. The occurrence of activating mutations in the gene encoding FMS-like tyrosine kinase 3 (FLT3) in patients with acute myeloid leukaemia (AML) has rendered inhibition of this receptor a promising therapeutic target. Due to possibility of cross-reactivity, we investigated the effect of the irreversible pan-ERBB inhibitor canertinib (CI-1033) on leukaemic cells expressing FLT3. The drug had anti-proliferative and apoptotic effects on primary AML cells and human leukaemic cell lines expressing mutated FLT3. In several AML patient samples, a blast cell population expressing FLT3-internal tandem duplication (ITD) was eradicated by canertinib. Canertinib inhibited receptor autophosphorylation and kinase activity of both mutated and FLT3 ligand stimulated wildtype FLT3, leading to inhibition of the PI3-kinase and MAP kinase pathways. Apoptotic induction was dependent on pro-apoptotic BH3-only protein BCL2L11/BIM because siRNA silencing attenuated apoptosis. Moreover, the drug induced regression of cells expressing FLT3-ITD in a murine in vivo-transplantation model at previously described tolerated doses. These results indicate that canertinib, as an irreversible TKI, could constitute a novel treatment regimen in patients with mutated or overexpressed FLT3., (© 2011 Blackwell Publishing Ltd.)

Canertinib is a novel ErbB-receptor inhibitor currently in clinical development for the treatment of solid tumors overexpressing ErbB-receptors. We have recently demonstrated that canertinib displays anti-proliferative and pro-apoptotic effects in human myeloid leukemia cells devoid of ErbB-receptors. The mechanism mediating these effects are however unknown. In this study, we show that canertinib is able to act as a multi-kinase inhibitor by inhibition of several intracellular kinases involved in T-cell signaling such as Akt, Erk1/2 and Zap-70, and reduced Lck protein expression in the human T-cell leukemia cell line Jurkat. Treatment with canertinib at a concentration of 2 μM caused accumulation of Jurkat cells in the G(1) cell cycle phase and increased doses induced apoptosis in a time-dependent manner. Apoptotic signs of treated cells were detected by Annexin V staining and cleavage of PARP, caspase-3, -8, -9, -10 and Bid. A subset of the pro-apoptotic signals mediated by canertinib could be significantly reduced by specific caspase inhibitors. Taken together, these results demonstrate the dual ability of canertinib to downregulate important signaling pathways and to activate caspase-mediated intrinsic apoptosis pathway in human T-cell leukemia cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Epidermal growth factor (EGF) receptor tyrosine kinase inhibitors have recently been shown to display anti-neoplastic effects in human malignant myeloid cells. Our study was initiated in order to determine the effect of the pan-ErbB receptor tyrosine kinase inhibitor, canertinib (CI-1033), on growth and survival of human leukemia (HL-60 and U-937) cells. We show that treatment of HL-60 and U-937 cells with canertinib significantly inhibits growth of both cell lines in a dose-dependent manner; half maximal effective dose (IC(50)) in HL-60 and U-937 cells was approximately 2.5 microM and 1.0 microM, respectively. Treatment with 2 microM canertinib promoted a G(1) cell cycle arrest, whereas doses of 5 microM or more induced apoptosis as determined by the Annexin V method and cleavage of poly-(ADP-ribose) polymerase (PARP). HL-60 and U-937 cells lacked EGF-receptor transcript but expressed ErbB2-4 mRNA as determined by RT-PCR. However, none of the corresponding ErbB-receptor proteins could be detected by Western blot analysis. We conclude that canertinib induces apoptosis in HL-60 and U-937 cells devoid of functional ErbB1-4 receptors. Our results suggest that canertinib could be of potential clinical interest in the treatment of acute myeloid leukemia., (Copyright 2010 Elsevier Inc. All rights reserved.)

Members of the epidermal growth factor (EGF) family of structurally related tyrosine kinase receptors, known as the ErbB receptors (EGFR/ErbB1/HER1, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4) and their respective ligands, have been suggested to be involved in the development and progression of malignant melanoma. Here we investigate the effects of the ErbB1 tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) on human malignant melanoma cells (RaH3 and RaH5) in vitro. ZD1839 inhibited proliferation of exponentially growing RaH3 and RaH5 cells in a dose-dependent manner with a half-maximally effective dose of 3.5 and 2.0 micromol/l, respectively. Cell growth was inhibited at 0.1 micromol/l ZD1839 in both cell lines. Maximal inhibition was accomplished at 10 micromol/l ZD1839; however, the effect was not complete as both cell lines showed a continuous slow growth during the treatment period. Flow cytometry analysis of cell-cycle distribution showed that ZD1839 treatment caused accumulation of RaH3 and RaH5 cells in the G1 phase. The growth arrest induced by ZD1839 coincided with upregulation of the cyclin-dependent kinase inhibitor p27 KIP 1. There was no increase in apoptosis as determined by analysis of plasma phosphatidyl serine redistribution. Western blot analysis revealed that ZD1839 substantially reduced tyrosine phosphorylation of ErbB1 as well as ErbB2 and ErbB3. This was accompanied by a concomitant decrease in Akt-phosphorylation, Erk1/2-phosphorylation, and Stat3-phosphorylation. Our results show that ZD1839 interferes with the growth of human malignant melanoma cells by cytostatic effects. These findings indicate the possible use of ErbB receptor kinase inhibitors as a novel treatment strategy in malignant melanoma.

Objective: Cell types present in synovial joint tissues and during synovitis are known to produce epidermal growth factor receptor (EGFR)/ErbB-1/HER-1 and the potent EGFR-ligand transforming growth factor-alpha (TGF-alpha) in vitro. Concomitant expression of TGF-alpha, EGFR, and ErbB2 gives a strong proliferative drive in vitro and in vivo. However, the presence of TGF-alpha and members of the EGFR/EGFR-ligand family has not been thoroughly investigated in joint tissue in vivo. We aimed to determine whether TGF-alpha, EGFR, and ErbB2 are present in human synovial joints, especially during rheumatoid arthritis (RA)., Methods: TGF-alpha protein was immunodetected in knee synovial fluid (SF) collected from 23 RA patients, eight patients with other arthritic conditions, two osteoarthritis (OA) patients, and six post-traumatic patients (control). TGF-alpha mRNA and TGF-alpha, ErbB2, EGFR, and CD68 immunoreactivity were detected in knee synovial biopsies (6 RA/2 OA/6 control) using in situ hybridization and immunohistochemistry. TGF-alpha mRNA was determined in SF cells by reverse transcription polymerase chain reaction (RT-PCR) and/or the Northern blot technique., Results: TGF-alpha protein was found in the synovial membrane (SM) and in the majority of SF samples. TGF-alpha levels were significantly higher (p < 0.001) in SF of RA patients than controls, TGF-alpha protein and mRNA were increased and more widespread in SM of RA patients. In addition, white blood cells collected from RA SF expressed TGF-alpha mRNA. Immunoreactivity for ErbB2 was found in SM and was more widespread in RA patients than in controls., Conclusion: The presence of TGF-alpha in normal SF and SM may indicate a physiological maintenance function. The increased expression of TGF-alpha and ErbB2 in RA SF and SM may give rise to an abnormal growth pattern, contributing to inflammatory synovial hyperplasia.

Induction of G(1) arrest by TGF-beta correlates with the regulation of p21(Cip1) and p27(Kip1), members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-beta(1)-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-beta is diminished in T cells from mice deficient for both p21(Cip1) and p27(Kip1) (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8(+) T cells from DKO mice, TGF-beta is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15(Ink4b) in T cells stimulated in the presence of TGF-beta is not essential, as TGF-beta also efficiently suppressed proliferation of T cells from p15(Ink4b-/-) mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-beta, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3(-/-) T cells to the induction of growth arrest by TGF-beta. In summary, the growth suppressive effects of TGF-beta in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-beta by lowering thresholds for a maximal mitogenic response.

Background: The receptors for transforming growth factor beta (TGF-beta) and their signaling intermediates make up an important tumor-suppressor pathway. The role of one of these intermediates--Smad3--in the pathogenesis of lymphoid neoplasia is unknown., Methods: We measured Smad3 messenger RNA (mRNA) and protein in leukemia cells obtained at diagnosis from 19 children with acute leukemia, including 10 with T-cell acute lymphoblastic leukemia (ALL), 7 with pre-B-cell ALL, and 2 with acute nonlymphoblastic leukemia (ANLL). All nine exons of the SMAD3 gene (MADH3) were sequenced. Mice in which one or both alleles of Smad3 were inactivated were used to evaluate the role of Smad3 in the response of normal T cells to TGF-beta and in the susceptibility to spontaneous leukemogenesis in mice in which both alleles of the tumor suppressor p27Kip1 were deleted., Results: Smad3 protein was absent in T-cell ALL but present in pre-B-cell ALL and ANLL. No mutations were found in the MADH3 gene in T-cell ALL, and Smad3 mRNA was present in T-cell ALL and normal T cells at similar levels. In mice, the loss of one allele for Smad3 impairs the inhibitory effect of TGF-beta on the proliferation of normal T cells and works in tandem with the homozygous inactivation of p27Kip1 to promote T-cell leukemogenesis., Conclusions: Loss of Smad3 protein is a specific feature of pediatric T-cell ALL. A reduction in Smad3 expression and the loss of p27Kip1 work synergistically to promote T-cell leukemogenesis in mice., (Copyright 2004 Massachusetts Medical Society)

Global gene expression analysis using microarrays has been used to characterize the molecular profile of tumors. Gene expression variability at the mRNA level can be caused by a number of different events, including novel signaling, downstream activation of transcription enhancers or silencers, somatic mutation, and genetic amplification or deletion. Genomic amplifications are commonly observed in cancer and often include known oncogenes. The tyrosine kinase-type cell surface receptor, ERBB2, is an oncogene located on chromosome 17q21.1 that is amplified in 10-40% of breast tumors. We report for the first time that phenylethanolamine N-methyltransferase (PNMT), proteasome subunit, beta type 3 (PSMB3), ribosomal protein L19 (RPL19), and nuclear receptor subfamily 1, group D, member 1 (NR1D1) are coexpressed with ERBB2 in 34 breast cancer biopsies and also mapped within the same chromosomal location as the ERBB2 gene. Consistent with previous reports, we also observed that the steroidogenic acute regulatory protein-related gene, MLN64, and growth factor receptor bound protein 7 were coexpressed with ERBB2. Coexpression and colocalization of PNMT and MLN64 with ERBB2 suggested that the amplification of ERBB2 includes the chromosomal region harboring these genes. This hypothesis was validated in a subset of 12 biopsies. Gene amplification of ERBB2, PNMT, and MLN64 significantly correlated with increased mRNA gene expression (P < 0.05). These results suggest that gene expression profiling of breast biopsies may become a valuable method for adequately characterizing and choosing treatment modality for patients with breast cancer.

We have previously reported that the human monocytoid cell line U-937-1 constitutively expresses transforming growth factor-alpha (TGF-alpha) and that the steady-state levels of TGF-alpha mRNA as well as TGF-alpha protein release increase when U-937-1 cells are differentiated towards monocytes/macrophages. Interleukin-6 (IL-6), which has been shown to have growth-stimulatory effects on a number of cell types, has recently been shown to enhance TGF-alpha expression in keratinocytes. In the present study we investigated whether TGF-alpha expression in macrophage-like cells could be regulated by IL-6 using U-937-1 cells as a model system of monocyte/macrophage differentiation. U-937-1 cells were differentiated with retinoic acid (RA), vitamin D3 (Vit-D3) or phorbol-12-myristate-13-acetate (PMA) for 4 days and were then treated with human recombinant IL-6 (1000 IU/ml) for up to 24 hr. Northern blot analysis revealed that cells differentiated with PMA, inducing the phenotype of a secretory macrophage, markedly increased their TGF-alpha mRNA levels (2.7-fold) when treated with IL-6; the response was maximal at 6 hr and remained high at 12 hr. The expression of the TGF-alpha gene was accompanied by release of TGF-alpha protein into the cell culture medium, irrespective of differentiating agent, as demonstrated by enzyme-linked immunosorbent assay (ELISA), as well as by surface expression of pro-TGF-alpha as determined by indirect immunofluorescent cytometry. However, the superinduction of the TGF-alpha gene by IL-6 in cells differentiated with PMA was not accompanied by any increase in TGF-alpha protein release or pro-TGF-alpha surface expression. We conclude that since IL-6 causes increased steady-state levels of TGF-alpha mRNA in macrophage-like cells, it may prime these cells for production of this growth factor. Furthermore, we have shown that the IL-6 receptor complex is functional in U-937-1 cells induced to differentiate towards a secretory macrophage by treatment with PMA.

The estrogen receptor plays a critical role in the pathogenesis and clinical behavior of breast cancer. To better understand the molecular basis of estrogen-dependent forms of this disease we studied gene expression profiles from 53 primary breast cancer biopsies. Gene expression data for more than 7000 genes were generated from each tumor sample with oligo microarrays. A standard correlation-clustering algorithm identified 18 genes that co-clustered with estrogen receptor alpha. Eleven of these genes had previously been associated with estrogen regulation or breast tumorigenesis including trefoil factor 1 and estrogen regulated LIV-1. Additional study of these 18 genes may further delineate the role of estrogen receptor in breast cancer, generate new predictive biomarkers for response to endocrine therapies and identify novel therapeutic targets.

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

The pattern of susceptibility of malignant cells to the cytostatic drug suramin is not fully clarified. Therefore, in the present paper we have assessed the effects of suramin on the growth of eight cell lines derived from human malignant fibrous histiocytomas, by measuring DNA synthesis. The effect of suramin (10-200 microg/ml) on cells either unstimulated, or stimulated with platelet-derived growth factor (PDGF)-AA (10 ng/ml), PDGF-BB (10 ng/ml) or 10% fetal calf serum was studied. Four out of five cell lines unable to thrive without external growth factors showed growth inhibition by suramin. The two cell lines able to grow under serum-free conditions were unaffected by high-dose suramin. The exposure to suramin, at 200 microg/ml, abolished the growth stimulation caused by PDGF-AA and -BB. In contrast, a low dose of suramin (50 microg/ml), with or without PDGF, caused growth-stimulating effects in some cell lines. Our results indicate that high doses of suramin inhibit growth of malignant fibrous histiocytomas in vitro and that suramin exerts its growth-inhibitory effects on cells dependent on external growth factors. Low-dose treatment with suramin, however, may instead promote growth in both serum-dependent and -independent tumor cell lines.

Platelet-derived growth factor (PDGF) has been proposed to play an important role in the growth of tumors. In order to study the effects of PDGF-AB on tumor growth in vivo, sarcoma-bearing mice were treated with PDGF-AB. The tumors, a malignant fibrous histiocytoma and an osteosarcoma, had functional PDGF receptors in vitro, as demonstrated by stimulation of PDGF-AB using a [3H]thymidine incorporation assay. Immunohistochemistry also revealed that both sarcoma xenografts expressed PDGF receptors. The tumor-bearing mice were given human PDGF-AB for 14 days, either continuously by an intraperitoneally placed mini-osmotic pump, or by daily injections. No effects on tumor growth in vivo were observed, as measured by tumor volume, autoradiography or cell cycle distribution. The histological appearance and ploidy of the tumors remained unaltered. The results indicate that, although the tumor cells are stimulated by PDGF-AB in vitro, the in vivo milieu or tumor growth pattern may render the tumors less susceptible to exogenously administered PDGF-AB in vivo.

Eight human malignant fibrous histiocytomas were examined in vitro, in order to relate their growth properties to mRNA expression for platelet-derived growth factor (PDGF), PDGF receptor (PDGF-R), transforming growth factor-alpha (TGF-alpha) and the epidermal growth factor receptor (EGF-R). Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that all cell lines expressed mRNA for PDGF-R alpha and/or PDGF-R beta; six cell lines expressed mRNA for the PDGF-A chain, with one cell line coexpressing PDGF-B chain mRNA; seven cell lines expressed mRNA for TGF-alpha whereas six cell lines expressed EGF-R mRNA. Conditioned medium from three cell lines contained PDGF; none of the cell lines released TGF-alpha. Two cell lines grew without serum requirements; whereas both expressed mRNA for PDGF, PDGF-R, TGF-alpha and EGF-R, other cell lines, unable to grow without serum, showed the same combination of growth factor/growth factor receptor expression. The two cell lines able to grow without serum were also shown to be stimulated by the addition of PDGF-BB. These findings show that simultaneous expression of mRNA for a growth factor and its receptor does not necessarily imply an autocrine or paracrine loop. However, two of our cell lines fulfil the requirements of possible PDGF-related autocrine and paracrine regulation.

The kinetics of exogenously given 125I-platelet-derived growth factor-AB (PDGF-AB) was studied in mice. 125I-PDGF-AB was injected either intraperitoneally, intramuscularly or subcutaneously and the resulting concentrations of 125I-radioactivity monitored in the blood at different times. The serum levels of 125I-radioactivity rose to a maximum 2-4 hours after injection, before decreasing. Precipitation of serum with trichloroacetic acid demonstrated that 50 per cent or more of the 125I remained in macromolecular form. Further, gel chromatography studies showed that the molecular size of the labelled material in serum, three hours after injection, was the same as that of the original 125I-PDGF-AB. In addition, a low-molecular weight fraction was observed, indicating the presence of degradation products. The largest proportion of degraded material was obtained after subcutaneous administration. The absence of partially degraded 125I-labelled fragments, e.g. 125I oligopeptides, indicates complete rather than limited degradation and suggests that the 125I-PDGF-AB had been processed by cellular uptake. It is concluded that extra-vascularly given PDGF-AB in mice is taken up into the blood in intact macromolecular form. This finding suggests that it is possible to administer PDGF extravascularly to obtain a prolonged increase in the concentration of intact PDGF in the blood.

Eosinophils have a characteristic content of cationic proteins, stored in core-containing specific granules and released at sites of inflammation; coreless granules (sometimes called primary) are present in eosinophil promyelocytes. In order to determine a possible relationship between the two granule subsets, immunoelectron-microscopic techniques were used to determine the presence and precise intragranular distribution of major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and arylsulfatase B of eosinophil granules, as well as the Charcot-Leyden crystal (CLC) protein, in eosinophil progenitors of the bone marrow. MBP, ECP, EPO, and arylsulfatase B were observed in both coreless and core-containing (specific) granules. The difference in the distribution of MBP, having a uniform distribution in coreless granules and a crystalloid distribution in core-containing (specific) granules, could indicate a maturational process of a common organelle. CLC protein was distributed in the cytosol, in the euchromatin of the nuclei, but was also present in a rare granular compartment of both immature and mature eosinophils. The present findings suggest that coreless granules develop into core-containing specific granules.

We have previously demonstrated that human promyelocytic HL-60 cells express transforming growth factor-alpha (TGF-alpha) during granulocytic differentiation. The present experiments were carried out in order to determine whether cells differentiated towards monocytes/macrophages will analogously express the TGF-alpha proto-oncogene product. HL-60 cells were induced to differentiate with 1 microM 1,alpha 25-dihydroxycholecalciferol (vitamin D3), and the human monocytoid cell line, U-937, was induced with 1 microM retinoic acid (RA), 0.1 microM vitamin D3, or 0.16 microM phorbol-12-myristate-13-acetate (PMA), ie experimental protocols known to induce monocyte/macrophage differentiation in these cells. In HL-60 cells, lacking constitutive TGF-alpha mRNA, vitamin D3 caused expression of the TGF-alpha gene and protein as demonstrated by Northern blot analysis and enzyme-linked immunoabsorbant assay (ELISA). In U-937 cells, showing constitutive TGF-alpha expression, RA but not vitamin D3 or PMA, caused marked increase in TGF-alpha mRNA (approximately 5-fold) and protein (approximately 3-fold) levels. In both cell lines the increase in TGF-alpha mRNA was evident within 24 h and continued throughout the observation period. Thus, it is established that differentiation of human leukemia cells towards monocytes/macrophages may be accompanied by TGF-alpha gene and protein expression in vitro. This is in conformity with the observed ability of mature activated macrophages to produce TGF-alpha.

We have previously demonstrated a constitutive expression of transforming growth factor alpha (TGF-alpha) in normal human blood eosinophils, both at the mRNA and protein level. This may indicate a novel function of the eosinophil, the regulation of which has not been clarified. Therefore human white blood cells (WBC) were treated with potential regulators of eosinophil function. Northern blot analysis demonstrated that human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) caused a time and dose-dependent 2- to 3-fold increase of TGF-alpha mRNA levels, in relation to incubation in the absence of cytokine; maximal response was attained within 4 h of incubation. In contrast, IL-5 failed to influence the expression of the TGF-alpha gene. In situ analysis of GM-CSF- or IL-3-stimulated cells showed that eosinophils remained the sole cell type expressing TGF-alpha mRNA. However, whereas GM-CSF significantly induced, within 1 h, release of immunoreactive TGF-alpha protein, IL-3 was insufficient in this respect. In conclusion, our findings indicate that expression of TGF-alpha gene and protein in normal blood eosinophils is differently regulated by GM-CSF and IL-3.

Transforming growth factor alpha (TGF-alpha) is a pleiotropic factor mediating numerous cellular responses in normal and transformed cells. This includes differentiation, proliferation, migration, and formation of extracellular matrix. TGF-alpha has been demonstrated in circulating eosinophils from the idiopathic hypereosinophilic syndrome and in differentiating promyelocytic leukemia cells in vitro. Whether TGF-alpha production also occurs in normal human blood cells is not known. Northern blot analysis showed that normal human white blood cells consistently expressed the TGF-alpha gene in 47 out of 47 donors. Cell preparations enriched in mononucleated cells, and devoid of granulocytes, showed no TGF-alpha mRNA. In situ hybridization experiments assigned the TGF-alpha gene expression to the eosinophils; 100% of the eosinophils and no other cell types were specifically recognized by the complementary human TGF-alpha riboprobe. White blood cells, incubated at 37 degrees C for up to 6 hours, released immunoreactive TGF-alpha to the incubation medium, as determined by ELISA. In contrast, no TGF-alpha protein was detected in the incubation medium of mononuclear cells. It is concluded that TGF-alpha is constitutively produced and released by normal human blood eosinophils. TGF-alpha provided by eosinophils, may participate in the inflammatory reaction by interacting with mesenchymal and epithelial cells, thus promoting fibrosis or neovascularization.

The process of myeloid differentiation in human promyelocytic leukemia cells (HL-60) is accompanied by the coordinate expression of numerous protooncogenes. To investigate the expression of transforming growth factor alpha (TGF-alpha) in myeloid differentiation, HL-60 cells were induced to differentiate into granulocytes with 1.25% dimethyl sulfoxide, 0.2 microM all-trans retinoic acid, or 500 microM N6,O2-dibutyryladenosine-3'5'-cyclic monophosphate or differentiated along the monocyte/macrophage pathway with 0.1 microM phorbol-12-myristate-13-acetate. Using Northern blot analyses, TGF-alpha transcripts were detected within 24 h of treatment in cells differentiating toward granulocytes; maximal levels of gene expression were reached after 3 days or later and remained essentially constant throughout the observation period. These cells released TGF-alpha protein, as demonstrated by analysis of the incubation medium. In contrast, no TGF-alpha RNA or protein was detectable in HL-60 cell cultures when induced with phorbol-12-myristate-13-acetate. Epidermal growth factor receptor transcripts could not be detected either in undifferentiated or in differentiated HL-60 cells; therefore it appears as if an autocrine loop involving TGF-alpha in HL-60 cells is unlikely. In conclusion, the results demonstrate, for the first time, the expression of TGF-alpha in human granulocyte precursor cells. Our findings may indicate novel regulatory pathways in hematopoiesis.

The effect of suramin on tumor growth and morphology in two different human osteosarcoma xenografts (L-I OSM and L-II OSM) grown in BALB/cA-nu/nu mice was studied. Suramin (total dose, 720 mg/kg) given by i.p. injection (60 mg/kg/dose) for up to 9 weeks significantly inhibited osteosarcoma cell growth in both tumors, suramin-treated tumors showing only one-third or less of the volume of nontreated controls. Cell cycle distribution of tumor cells measured by DNA flow cytometry demonstrated that suramin treated caused accumulation of cells in the S and G2 phases of the cell cycle, in both L-I OSM and L-II OSM. In the aneuploid L-II OSM tumor suramin preferentially inhibited the growth of aneuploid cells, leading to a decrease in the ratio of aneuploid to diploid cells. Both osteosarcomas retained their histological appearance and the liver, spleen, heart, and kidneys of the treated animals were unaffected by suramin. These results are compatible with the view that suramin inhibits the growth of human osteosarcomas by cytostatic effects.