Retroviral Expression of Transforming Growth Factor-Alpha Does Not Transform Fibroblasts or Keratinocytes

Transforming growth factor α (TGFα) is a peptide so named because it helps to impart anchorage-independent growth to normal rat kidney (NRK) cells in vitro and is secreted by many rodent and human tumor cells. To directly investigate the transforming properties of this factor, we constructed a replication-defective murine retrovirus that expresses the human sequence coding for TGFα. infection of NIH/3T3 cells with the TGFα retrovirus led to the integration of a transcriptionally active provirus and overexpression of biologically active TGFα, but failed to induce morphologic transformation. Similarly, the TGFα retrovirus failed to induce morphologic transformation of five other types of rodent fibroblasts.We also investigated the effect of TGFα expression on the growth of BALB/MK mouse keratinocytes, which require epidermal growth factor (EGF) for proliferation. We show that exogenously added TGFα is an extremely potent mitogen for BALB/MK cells. However, retroviral expression of TGFa in BALB/MK cells failed to relieve dependence on exogenously added EGF (or TGFα) for cell growth. These results suggest that overexpression of TGFα does not, by itself, transform rodent fibroblasts or keratinocytes

Identification of Serine 643 of Protein Kinase C-δ as an Important Autophosphorylation Site for Its Enzymatic Activity

To investigate the role of serine/threonine autophosphorylation of protein kinase C-delta (PKC-delta), we mutated serine 643 of PKC-delta to an alanine residue (PKC-deltaS643A). Two different expression vectors containing PKC-deltaS643A mutant cDNAs were transfected and expressed in 32D myeloid progenitor cells. In vitro autophosphorylation assays demonstrated 65-83% reduction in autophosphorylation of PKC-deltaS643A in comparison to wild type PKC-delta (PKC-deltaWT). The enzymatic activity of PKC-deltaS643A mutant as measured by phosphorylating the PKC-delta pseudosubstrate region-derived substrate was also reduced more than 70% in comparison to that of PKC-deltaWT. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that at least one phosphopeptide was absent in PKC-deltaS643A when compared with PKC-deltaWT, further substantiating that serine 643 is phosphorylated in vivo. Localization and 12-O-tetradecanoylphorbol-13-acetate-dependent translocation and tyrosine phosphorylation of PKC-deltaS643A were not altered in comparison to PKC-deltaWT, indicating that mutagenesis did not affect the structural integrity of the mutant protein. 12-O-Tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells overexpressing PKC-deltaS643A mutant protein was impaired in comparison to that of PKC-deltaWT transfectant. Taken together, our results demonstrate that serine 643 of PKC-delta is a major autophosphorylation site, and phosphorylation of this site plays an important role in controlling its enzymatic activity and biological function

The expression of the mouse VpreB/λ5 locus in transformed cell lines and tumors of the B lineage differentiation pathway

The expression of RNA transcripts from two pre B lymphocyte related genes, VpreB and λ5, has been studied in a series of transformed cell lines which appear frozen at different states of B lineage differentiation, from early progenitors to surface Ig positive B cells. In the HAFTL-1 cell line, which arose from fetal liver by transformation with a retrovlrus containing the Hras oncogene, Northern analysis of poly A+ mRNA as well as in situ hybridization of RNA In single cells revealed that λ5 and VpreB are already expressed at the progenitor stage and increase in expression as the progenitors differentiate to precursor (preB) cells, or are turned off as the progenitors differentiate to myeloid cells. Continued rearrangements of Ig genes in pre B cell lines leading to Ig expression on the surface of NFS-5 pre B cells do not influence the continued expression of VpreB and λ5. Surface Ig-positive B lineage cell lines also express the pre B-related genes. Both Ly1+ as well as Ly1− pre B cells are VpreB and λ5positlve. Lipopolysaccharide (LPS) stimulation of 70Z/3 pre B cells does not turn off λ5 expression. It therefore appears that, at least In transformed cell lines, the expression of VpreB and λ5, is not directly regulated by the expression of μH, κL, or λL chains, LPS reactivity, or the Ly1 surface antigen. Fusion of plasmacytoma cells with normal pre B cells to generate pre B hybridomas leads to down-regulation of VpreB/λ5 expression. These results suggest that different trans-acting factors in more mature cells might down-regulate the expression of VpreB/λ

Retroviral Expression of Transforming Growth Factor-Alpha Does Not Transform Fibroblasts or Keratinocytes

Transforming growth factor α (TGFα) is a peptide so named because it helps to impart anchorage-independent growth to normal rat kidney (NRK) cells in vitro and is secreted by many rodent and human tumor cells. To directly investigate the transforming properties of this factor, we constructed a replication-defective murine retrovirus that expresses the human sequence coding for TGFα. infection of NIH/3T3 cells with the TGFα retrovirus led to the integration of a transcriptionally active provirus and overexpression of biologically active TGFα, but failed to induce morphologic transformation. Similarly, the TGFα retrovirus failed to induce morphologic transformation of five other types of rodent fibroblasts.We also investigated the effect of TGFα expression on the growth of BALB/MK mouse keratinocytes, which require epidermal growth factor (EGF) for proliferation. We show that exogenously added TGFα is an extremely potent mitogen for BALB/MK cells. However, retroviral expression of TGFa in BALB/MK cells failed to relieve dependence on exogenously added EGF (or TGFα) for cell growth. These results suggest that overexpression of TGFα does not, by itself, transform rodent fibroblasts or keratinocytes

Cell-cell adhesion mediated by binding of membrane-anchored transforming growth factor alpha to epidermal growth factor receptors promotes cell proliferation

The precursor for transforming growth factor alpha, pro-TGF-alpha, is a cell surface glycoprotein that can establish contact with epidermal growth factor (EGF) receptors on adjacent cells. To examine whether the pro-TGF-alpha/EGF receptor pair can simultaneously mediate cell adhesion and promote cell proliferation, we have expressed pro-TGF-alpha in a bone marrow stromal cell line. Expression of pro-TGF-alpha allows these cells to support long-term attachment of an EGF/interleukin-3-dependent hematopoietic progenitor cell line that expresses EGF receptors but is unable to adhere to normal stroma. This interaction is inhibited by soluble EGF receptor ligands. Further, the hematopoietic progenitor cells replicate their DNA while they are attached to the stromal cell layer and become foci of sustained cell proliferation. Thus, pro-TGF-alpha and the EGF receptor can function as mediators of intercellular adhesion and this interaction may promote a mitogenic response. We propose the term juxtacrine to designate this form of stimulation between adjacent cells

Accumulation of Fatty Alcohol in MCF-7 Breast Cancer Cells

The MCF-7 cell line (human breast epithelial cells) accumulates fatty alcohols. The fatty alcohols were identified as C16:0, C18:0, and C18:1 alcohols by thin-layer chromatography and gas chromatography/mass spectrometry. This accumulation of alcohols in MCF-7 was found in cultures of MCF-7 cells obtained from other laboratories but not in a variety of unrelated cell lines. The presence of the alcohols suggested an aberrant ether lipid metabolism in the MCF-7 cells. Therefore, the capacity for ether lipid biosynthesis was evaluated using cells incubated with either [14C]stearyl alcohol or [14C]stearic acid. MCF-7 cells incorporated less than 0.4% of the [14C]alcohol into ether-linked phospholipids, whereas the AB589 breast epithelial cells, used as a normal control for comparisons, did not accumulate fatty alcohol and incorporated approximately 20% of the radiolabeled alcohol into phospholipids containing ether linkages. Although the MCF-7 cells were unable to effectively incorporate the fatty alcohol into ether linkages, the cells were able to oxidize the alcohol to fatty acid. When incubated with [14C]stearic acid, the conversion to radiolabeled fatty alcohol in MCF-7 cells was approximately four times higher than the alcohol levels found in AB589 cells. While deficient in the ability to synthesize ether linkages, the MCF-7 cells did incorporate radiolabeled hexadecylglycerol, a precursor containing an ether linkage, into phospholipids. Collectively, the data indicate that the MCF-7 cells possess a deficiency in the alkyl DHAP synthase activity. A near absence of ether-linked lipids in the MCF-7 cells was indicated by the radiolabeling studies, and this finding was corroborated by results from HPLC analysis. Analyses of the partial glycerides, obtained from the enzymatic hydrolysis of cellular phospholipids, found only trace levels of ether lipids in the MCF-7 cells. The aberration in ether lipid biosynthesis did not correlate with the expression of the multidrug resistance phenotype in a series multidrug resistant MCF-7 variants. The results are discussed relative to the use of the MCF-7 cells as a model for investigations of ether lipid biosynthesis and the cellular physiology of ether lipids