Resistance to apoptotic cell death in a drug resistant T cell leukaemia cell line.

In this study, we investigated the responses of the T cell leukaemia cell line, CCRF-CEM, and a vincristine-resistant subline, CEM/VCR R, to the induction of cell death by serum withdrawal. This treatment was used to overcome any contribution of P-glycoprotein-mediated drug resistance to the responses of the CEM/VCR R cells. Following serum withdrawal both cell lines exhibited typical apoptotic responses including morphological changes and nucleosomal cleavage of the DNA. However, using several different assays for cell death the CEM/VCR R cell line was shown to undergo apoptosis at a slower rate than the parental CCRF-CEM cell line. Expression of c-Myc, Bcl-2 and p53 was found to be similar in both cell lines, discounting involvement of these proteins in the observed difference in apoptotic response. Given our previous finding that reorganisation of tubulin is involved in apoptosis, we examined the expression of alpha-, beta- and acetylated alpha-tubulin in the parental and resistant lines. The CEM/VCR R cell line had altered tubulin expression when compared to that of the CCRF/CEM line. Transnuclear microtubule networks were observed in log phase CEM/VCR R cells. In addition, increased expression of the acetylated form of the alpha-tubulin isotype suggested that a more stable microtubule network was present in the CEM/VCR R cells. These findings imply that the drug-resistance phenotype in the CEM/VCR R cells may involve the suppression of apoptosis, and that the development of an altered microtubule network may contribute to this suppression.

Multiple centrosomal microtubule organising centres and increased microtubule stability are early features of VP-16-induced apoptosis in CCRF-CEM cells.

Microtubular reorganisation contributing to apoptotic morphology occurs in normal and neoplastic cells undergoing apoptosis induced by cytotoxic drugs [1-3]. The aim of this study was to correlate the changes in the microtubules (MTs) with behavior of the centrosome in apoptotic cells, and to see whether post-translational changes in tubulin occurred with the emergence of apoptotic MT bands. Apoptosis was induced in the human T-cell leukaemia line (CCRF-CEM) by treatment with 17 microM etoposide over a 4 h period. The time course of changes was assessed using flow cytometry (FCM) and immunocytochemistry in cells labelled for a centrosomal antigen (CSP-alpha) or alpha-tubulins. One hour following treatment we observed multiple centrosomal microtubule organising centres (MTOCs) associated with the nucleus and the transient appearance of a subset of stable MTs detected with an antibody specific for acetylated alpha-tubulin, as the bands of MTs which lobulate the nucleus are formed. The altered properties of the MTs thus reflect changes in function as apoptosis progresses.

Increased tubulin acetylation accompanies reversion to stable ploidy in vincristine-resistant CCRF-CEM cells.

The T-cell leukemia line CCRF-CEM is unstable with respect to ploidy, whereas a vincristine-resistant subline, CEM/VCR R, maintains a stable pseudodiploid karyotype. Ploidy change in the parental cells requires the involvement of two cell cycle lesions. The first, in mitosis, prevents cell division after S-phase. The second, in G1, allows a cell with 4N DNA content to re-enter S-phase. We examined differences in expression of tubulin, a major component of the mitotic spindle and the cellular target for vincristine, between the two cell lines. Levels of the beta III isotype were decreased and levels of acetylated alpha-tubulin, a marker for microtubule stability, were increased in the CEM/VCR R cells relative to the parental line, which suggests that the CEM/VCR R cells have a more stable mitotic spindle. Both cell lines exhibit some level of constitutive expression of p53 and c-myc. Constitutive expression of and mutant p53 would contribute to the failure of these cells to recognise G1 checkpoints. Therefore, G1 checkpoint failure and the intrinsically less stable mitotic spindle in the CCRF-CEM cells may contribute to the observed ploidy instability. Conversely, the presence of markers of microtubule stability in the CEM/VCR R cells would predispose them to maintain their ploidy.

Integrin-matrix interactions affect the form of the structures developing from human mammary epithelial cells in collagen or fibrin gels.

The HB2 cell line, developed from luminal epithelial cells cultured from milk, forms ball-like structures in collagen gels which show a uniform branching response to hepatocyte growth factor. The alpha2beta1 integrin is the major integrin expressed by luminal epithelial cells, and the role of this integrin in mammary morphogenesis has been analysed using HB2 cells cultured in collagen gels and antibodies which affect integrin function. Selectivity of response was followed by comparing effects on morphogenesis in fibrin, where the alphavbeta1 integrin interacts with the matrix. In the presence of hepatocyte growth factor, using alpha2 and beta1 antibodies in collagen and alphav and beta1 antibodies in fibrin, complete blocking of the cell-matrix interaction inhibits cell survival. With partial blocking of the integrin-ligand interaction, the cells proliferate but form dissociated colonies. Activating antibodies to the beta1 integrin subunit which enhance the matrix interaction dramatically inhibit the branching and motility responses to hepatocyte growth factor. A series of non-blocking alpha2 reactive antibodies also inhibit these responses specifically in or on collagen. Studies with ras-transfected HB2 cells emphasise the importance of the alpha2beta1 collagen interaction in the development of form since HB2ras cells, which express reduced levels of the alpha2beta1 integrin, form dissociated colonies in collagen but not in fibrin. Treatment of HB2ras cells with a beta1 activating antibody, however, induces the formation of compact colonies. Even though the ras-transformants form colonies in agar, complete blocking of the alpha2beta1/collagen interaction does not allow survival in collagen. The results indicate that in mammary morphogenesis, the strength of the interaction of integrins with the extracellular matrix modulates the response to motogenic factors and contributes to the definition of form.

Breast tumour cell-induced down-regulation of type I collagen mRNA in fibroblasts.

This study investigated the modulation of type I collagen gene expression in normal fibroblasts by breast tumour cells. Northern analysis of total RNA extracted from stages I, II and III breast tumour tissue revealed that collagen mRNA levels were elevated in stage I tumours compared to the adjacent normal breast tissues, whereas they were decreased in stages II and III breast tumours. This aberrant collagen gene expression was confirmed by non-radioactive RNA:RNA in situ hybridization analysis of 30 breast carcinomas which localized the production of type I collagen mRNA to the stromal fibroblasts within the vicinity of the tumour cells. In order to determine whether the tumour cells were directly responsible for this altered collagen production by the adjacent fibroblasts, breast tumour cell lines were co-cultured with normal fibroblasts for in vitro assessment of collagen and steady-state collagen RNA levels. Co-culture of tumour cells and normal fibroblasts in the same dish resulted in down-regulation of collagen mRNA and protein. Treatment of the fibroblasts with tumour-cell conditioned medium also resulted in decreased collagen protein levels but the mRNA levels, however, remained unaltered. These results suggested that the tumour cells either secrete a labile 'factor', or express a cell surface protein requiring direct contact with the fibroblasts, resulting in down-regulation of collagen gene expression. Modulation of the ECM is a common characteristic of invading tumour cells and usually involves increased production of collagenases by the tumour cells or stromal fibroblasts. This study showed that tumour cells were also able to modulate collagen mRNA production by stromal fibroblasts, which may facilitate tumour cell invasion and metastasis.