Role of the cytoskeleton in laminin induced mammary gene expression

The differentiation of rat mammary epithelial cells is characterized both by morphologic changes and by the expression of a group of milk protein genes. We have previously shown that by culturing these cells on the basement membrane glycoprotein laminin, the synthesis of the milk proteins, transferrin, Α-casein, and Α-lactalbumin is induced. In order to determine if this effect is mediated through the cytoskeleton, we have treated these cells with cytochalasin D and colchicine. Treatment with cytochalasin D or colchicine for 24 h inhibits the accumulation of Α-casein, transferrin, and Α-lactalbumin without significant effect on general protein synthesis. Pulse chase studies show that cytochalasin D does not alter the intracellular turnover of Α-casein or transferrin. Additionally, treatment with cytochalasin D causes an early (within 1 h) increase in secretion of Α-casein and transferrin suggesting that the actin cytoskeleton provides a meshwork for secretory vesicles. The disruption of this network enhances the secretion of preformed proteins. However, long term (24 h) treatment with cytochalasin D inhibits synthesis of these milk proteins. Northern blot analysis indicates that treatment with cytochalasin D or colchicine inhibits the laminin induced increase in Α-casein, Α-lactalbumin, and transferrin mRNAs. These studies indicate that the major effect of the cytoskeleton on laminin induced milk protein gene expression occurs at the level of accumulation of mRNAs for these proteins. We conclude that the expression of laminin induced milk protein gene expression in primary rat mammary cultures depends on the integrity of the actin and microtubule cytoskeleton.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49876/1/1041350103_ftp.pd

Clustering of cell surface laminin enhances its association with the cytoskeleton

In order to provide evidence for an association of cell surface laminin with the cytoskeleton, we have examined the detergent extractability of cell surface laminin on murine fibrosarcoma cells. We utilized indirect immunofluorescence with affinity-purified antilaminin antibodies to determine the distribution, mobility and detergent extractability of laminin bound to the cell surface. We demonstrate that antibody induces clustering of cell surface laminin rendering it resistant to detergent extraction.At low receptor occupancy, approx. 80% of cell surface laminin is detergent-extractable. If cell surface laminin is induced to cluster with anti-laminin antibody, IB4 isolectin from Bandeiraea simplicifolia or by high receptor occupancy, then it is rendered resistant to detergent extraction. This process is temperature-sensitive and inhibited by cytochalasin D (CD). On the basis of these findings, we propose a model in which laminin anchored in the basement membrane in vivo affects the cellular cytoskeleton by facilitating the clustering of cell surface transmembrane laminin receptors which are able to interact with cellular actin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26112/1/0000188.pd

Interactions of the 67 kDa laminin receptor and its precursor with laminin

The 67LR (67 kDa laminin receptor) enables cells to interact with components of the extracellular matrix. The molecule is derived from the 37LRP (37 kDa laminin receptor precursor); however, the precise molecular mechanism of this conversion is unknown. Recombinant 37LRP, expressed in and purified from Escherichia coli, bound to human laminin in a SPR (surface plasmon resonance) experiment. 67LR isolated from human breast-cancer-derived cells in culture was also shown to bind to laminin by SPR. However, the kinetics of association are qualitatively different. 37LRP, but not 67LR, binds to heparan sulfate. The binding of 37LRP to heparan sulfate did not affect the interaction of 37LRP with laminin. In contrast, heparan sulfate reduces the extent of binding of laminin to 67LR. Taken together, these results show that 37LRP has some of the biological activities of 67LR, even prior to the conversion event. However, the conversion affects the sites of interaction with both laminin and heparan sulfate

Thrombospondin binding by human squamous carcinoma and melanoma cells: Relationship to biological activity

Human squamous carcinoma cells attach and spread on thrombospondin (TSP)-coated culture dishes but exhibit significant variability among individual cell lines in their degree of responsiveness. Using a highly responsive squamous carcinoma line and a cell line which is much less responsive (as well as a human melanoma cell line which does not respond at all in the adhesion assay), we have examined binding of exogenous radiolabeled TSP. The cells which were the most responsive to TSP in the adhesion assay bound the greatest amount of radiolabeled ligand. Binding was time- and dose-dependent, saturable, inhibitable with excess unlabeled TSP, reversible, and specific. The less-responsive squamous carcinoma cells bound only 25-30% of the amount of TSP bound by the highly responsive cells while the nonresponsive melanoma cells bound less than 10% of the amount bound by the highly responsive squamous carcinoma cells. Our previous studies (J. Varani et al. (1986) Exp. Cell Res.167, 376) have shown that the highly responsive squamous carcinoma cells also synthesized the greatest amount of TSP as indicated by biosynthetic labeling studies. The less-responsive squamous carcinoma cells were intermediate in synthetic activity and no synthetic activity was seen with the melanoma cells. These findings suggest that the amount of ligand bound may determine the degree of biological responsiveness and that endogenously synthesized TSP may be the source of that ligand.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27417/1/0000455.pd

Impact of opioid rescue medication for breakthrough pain on the efficacy and tolerability of long-acting opioids in patients with chronic non-malignant pain

There is little evidence that short-acting opioids as rescue medication for breakthrough pain is an optimal long-term treatment strategy in chronic non-malignant pain. We compared clinical studies of long-acting opioids that allowed short-acting opioid rescue medication with those that did not, to determine the impact of opioid rescue medication use on the analgesic efficacy and tolerability of chronic opioid therapy in patients with chronic non-malignant pain. We searched MEDLINE (1950 to July 2006) and EMBASE (1974 to July 2006) using terms for chronic non-malignant pain and long-acting opioids. Independent review of the search results identified 48 studies that met the study selection criteria. The effect of opioid rescue medication on analgesic efficacy and the incidence of common opioid-related side-effects were analysed using meta-regression. After adjusting for potentially confounding variables (study design and type of opioid), the difference in analgesic efficacy between the 'rescue' and the 'no rescue' studies was not significant, with regression coefficients close to 0 and 95% confidence intervals that excluded an effect of more than 18 points on a 0-100 scale in each case. There was also no significant difference between the 'rescue' and the 'no rescue' studies for the incidence of nausea, constipation, or somnolence in both the unadjusted and the adjusted analyses. We found no evidence that rescue medication with short-acting opioids for breakthrough pain affects analgesic efficacy of long-acting opioids or the incidence of common opioid-related side-effects among chronic non-malignant pain patients

Attachment, spreading and growth in vitro of highly malignant and low malignant murine fibrosarcoma cells

Highly malignant cell lines and low-malignant cell lines isolated from three different methylcholanthrene-induced murine fibrosarcomas were examined for their ability to attach to plastic dishes and collagen-coated dishes under serumfree conditions and in the presence of serum. Most of the cells from the three highly malignant lines attached and spread under all conditions. By 72h, there was a significant increase in the number of cells indicating that at least some of the cells had undergone division (even in the absence of serum). In contrast, fewer of the cells from the three low-malignant lines attached and spread on the plastic or collagen substrates in the absence of serum or in the presence of 0.1 per cent serum. However, when 15 μ g laminin per dish was added along with the lowmalignant cells, they then attached and spread on the plastic and collagen-coated dishes. Previous studies have indicated that the highly malignant lines express cell surface antigens that cross-react with laminin while the low-malignant cell lines do not. We speculate that the differences between the high- and low-malignant cells in the expression of cell surface laminin-like antigens contribute to the dissimilarities in attachment and spreading capacity. These differences may also contribute to the dissimilarity between these cells in malignant potential.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42592/1/10585_2005_Article_BF01758953.pd

Interactions Between Laminin Receptor and the Cytoskeleton During Translation and Cell Motility

Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration

Regulation of rat mammary gene expression by extracellular matrix components

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces [alpha]-casein and [alpha]-lactalbumin ([alpha]-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for [alpha]-, [beta]-, and [gamma]-caseins and [alpha]-LA. These studies demonstrated that ECM components induced [alpha]- and [beta]-caseins up to 10-fold and [alpha]-LA up to 3-fold, with no significant effect on [gamma]-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26485/1/0000021.pd