The p38 MAPK pathway mediates the growth inhibitory effects of interferon-alpha in BCR-ABL-expressing cells.

The mechanisms by which interferon-alpha (IFN-alpha) mediates its anti-leukemic effects in chronic myelogenous leukemia (CML) cells are not known. We determined whether p38 MAPK is activated by IFN-alpha in BCR-ABL-expressing cells and whether its function is required for the generation of growth inhibitory responses. IFN-alpha treatment induced phosphorylation/activation of p38 in the IFN-alpha-sensitive KT-1 cell line, but not in IFN-alpha-resistant K562 cells. Consistent with this, IFN-alpha treatment of KT-1 (but not K562) cells induced activation of the small GTPase Rac1, which functions as an upstream regulator of p38. In addition, IFN-alpha-dependent phosphorylation/activation of p38 was induced by treatment of primary granulocytes isolated from the peripheral blood of patients with CML. To define the functional role of the Rac1/p38 MAPK pathway in IFN-alpha signaling, the effects of pharmacological inhibition of p38 on the induction of IFN-alpha responses were determined. Treatment of KT-1 cells with the p38-specific inhibitors SB203580 and SB202190 reversed the growth inhibitory effects of IFN-alpha. On the other hand, the MEK kinase inhibitor PD098059 had no effects, further demonstrating the specificity of these findings. To directly determine the significance of IFN-alpha-dependent activation of p38 in the induction of the anti-leukemic effects of IFN-alpha, we evaluated the effects of p38 inhibition on leukemic colony formation in bone marrow samples of patients with CML. IFN-alpha inhibited leukemic granulocyte/macrophage colony formation in a dose-dependent manner, whereas concomitant treatment with p38 inhibitors reversed such an inhibition. Thus, the Rac1/p38 MAPK pathway is activated by IFN-alpha in BCR-ABL-expressing cells and appears to play a key role in the generation of the growth inhibitory effects of IFN-alpha in CML cells.

Engagement of the CrkL adaptor in interferon alpha signalling in BCR-ABL-expressing cells.

Interferon alpha (IFNalpha) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR-ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR-ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNalpha-signalling in the CML-derived KT-1 cell line, which expresses BCR-ABL and is sensitive to the growth inhibitory effects of IFNalpha. CrkL is constitutively associated with BCR-ABL in these cells and treatment with IFNalpha had no effect on the BCR-ABL/CrkL interaction. After IFNalpha stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNalpha-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL-Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the beta-casein promoter or the promoter of the PML gene, an IFNalpha-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR-ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNalpha in CML.

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to all-trans-retinoic acid.

Several signaling pathways are activated by all-trans-retinoic acid (RA) to mediate induction of differentiation and apoptosis of malignant cells. In the present study we provide evidence that the p38 MAP kinase pathway is activated in a RA-dependent manner in the NB-4, acute pro-myelocytic leukemia, and the MCF-7, breast carcinoma, cell lines. RA treatment of cells induces a time- and dose-dependent phosphorylation of p38, and such phosphorylation results in activation of its catalytic domain. p38 activation is not inducible by RA in a variant NB-4 cell line, NB-4.007/6, which is resistant to the effects of RA, suggesting a role for this pathway in the induction of RA responses. Our data also demonstrate that the small G-protein Rac1 is activated by RA and functions as an upstream regulator of p38 activation, whereas the MAPKAPK-2 serine kinase is a downstream effector for the RA-activated p38. To obtain information on the functional role of the Rac1/p38/MAPKAPK-2 pathway in RA signaling, the effects of pharmacological inhibition of p38 on RA-induced gene transcription and cell differentiation were determined. Our results indicate that treatment of cells with the SB203580 inhibitor does not inhibit RA-dependent gene transcription via retinoic acid response elements or induction of Stat1 protein expression. However, treatment with SB203580 or SB202190 strongly enhances RA-dependent induction of cell differentiation and RA-regulated growth inhibitory responses. Altogether, our findings demonstrate that the Rac1/p38 MAP kinase pathway is activated in a RA-dependent manner and exhibits negative regulatory effects on the induction of differentiation.

The Rac1/p38 mitogen-activated protein kinase pathway is required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins.

The p38 mitogen-activated protein (MAP) kinase is activated during engagement of the type I interferon (IFN) receptor and mediates signals essential for IFNalpha-dependent transcriptional activation via interferon-stimulated response elements without affecting formation of the ISGF3 complex. In the present study, we provide evidence that the small GTPase Rac1 is activated in a type I IFN-dependent manner and that its function is required for downstream engagement of the p38 MAP kinase pathway. We also demonstrate that p38 is required for IFNalpha-dependent gene transcription via GAS elements and regulates activation of the promoter of the PML gene that mediates growth inhibitory responses. In studies to determine whether the regulatory effects of p38 are mediated by serine phosphorylation of Stat1 or Stat3, we found that the p38 kinase inhibitors SB203580 or SB202190 or overexpression of a dominant negative p38 mutant do not inhibit phosphorylation of Stat1 or Stat3 on Ser-727 in several IFNalpha-sensitive cell lines. Altogether these data demonstrate that the Rac1/p38 MAP kinase signaling cascade plays a critical role in type I IFN signaling, functioning in cooperation with the Stat-pathway, to regulate transcriptional regulation of IFNalpha-sensitive genes and generation of growth inhibitory responses.

Treatment of medullary thyroid carcinoma by combined expression of suicide and interleukin-2 genes.

Inherited medullary thyroid carcinomas (MTC) are aggressive and resistant to conventional chemo- and radiotherapies. We evaluated a novel strategy for treatment of MTC, combining "suicide" and interleukin-2 (IL-2) gene therapies. Tumors were produced in Wag/Rij rats by orthotopic injection of the rMTC 6-23 cell line, and/or derivatives expressing the herpes simplex virus 1 thymidine kinase (HSV1-TK) gene (rMTC-TK). Ganciclovir, a nucleoside analog selectively transformed to a toxic metabolite by HSV1-TK, totally eradicated rMTC-TK tumors in 60% of the animals. 1:1 rMTC and rMTC-TK mixed tumors were also strongly inhibited by ganciclovir (P < 0.05), indicating the occurrence of an efficient "bystander" effect in vivo. Double labelling of rMTC cell membranes and apoptotic nuclei revealed that, as with the TK+ cells, some TK- cells died by apoptosis. A 1:1 mixture of rMTC and rMTC-TK cells was administered to produce established tumors and then rMTC cells, transfected to express the IL-2 gene (rMTC-IL2), were inoculated. Combined ganciclovir and IL-2 treatment improved the inhibition of tumor growth compared to that following ganciclovir alone (86% compared to 54%, P < 0.05). This treatment also significantly enhanced macrophage activation and tumor infiltration by CD8+ and CD4+ T lymphocytes. These results open an avenue for combining suicide and immunoregulatory gene therapies for MTC management in man.

Expression of laminin-2 by normal and neoplastic rat C cells during the development of medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT), their specific marker. C cells are located in contact with the basement membrane (BM) of the thyroid follicles, which is partly made up of the laminin-2 isoform synthesized by thyrocytes. During oncogenesis, proliferation of the C cells, invading the centre of the follicles, leads to a break in their normal contact with the BM. As specific interactions of cells with BM components, especially laminins, are important for proliferation and differentiation, we investigated the relationships of normal and neoplastic C cells with laminin in the Wag/Rij rat model of human MTC. Immunocytochemical studies showed a progressive loss of the laminin layer underlying the hyperplastic C cell nodules around the large dedifferentiated tumours. The alpha2, beta1 and gamma1 chains of the laminin-2 isoform were synthesized and secreted by rat MTC 6-23 cell cultures and the tumours induced by subcutaneous injection of these cells. In situ hybridization combined with anti-CT immunocytochemistry showed a low expression of alpha2 mRNA on differentiated C cells and thyrocytes, but an overexpression on immunonegative spontaneous MTC and induced intrathyroid tumours. The high level of alpha2 gene expression, together with tumour dedifferentiation, suggests a relationship with malignancy.

Influence of laminin substratum on cell proliferation and CALC I gene expression in medullary thyroid carcinoma C cell lines.

Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT) and CT gene-related peptide (CGRP), the two splice peptide products of the CALC I gene. Normal and hyperplastic C cells are intrafollicular, in contact with the basement membrane (BM) that is maintained around the differentiated tumors. To investigate the relationships between MTC evolution and BM constituents, we examined the modifications induced by laminin-1 and -2 (merosin), two isoforms colocalized in the follicular BM, on three MTC cell lines: murine rMTC 6-23 and CA-77 cells, and human TT cells. Laminin exerted a mitogenic activity on rMTC 6-23 and on TT cells, causing a concurrent decrease in both CT and CGRP mRNA levels and production of the peptides. Conversely, laminin reduced the proliferation rate and enhanced CGRP synthesis and secretion in CA-77 cells. This antiproliferative response, which coincides with an increase in differentiation markers, is comparable to that reported in normal cells and also in the neoplastic Caco-2 cell line. This suggests that laminin could exert opposite effects depending on the stage of tumor evolution.