The introduction and expression of genes in somatic cells is an innovative therapy for correcting genetic deficiency diseases and augmenting immune function. A potential obstacle to gene therapy is the elimination of such gene-modified cells by an immune response to novel protein products of the introduced genes. We are conducting an immunotherapy trial in which individuals seropositive for human immunodeficiency virus (HIV) receive CD8+ HIV-specific cytotoxic T cells modified by retroviral transduction to express a gene permitting positive and negative selection. However, five of six subjects developed cytotoxic T-lymphocyte responses specific for the novel protein and eliminated the transduced cytotoxic T cells. The rejection of genetically modified cells by these immunocompromised hosts suggests that strategies to render gene-modified cells less susceptible to host immune surveillance will be required for successful gene therapy of immunocompetent hosts.
The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56kk is phosphorylated in vivo at a carboxy-terminal tyrosine residue analogous to Tyr-527 of pp60`csrc. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56kk in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60`csrc, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56kk in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquistion of a posttranslationally activated version of pps6IckThe src gene family includes seven closely related sequences (fgr, fyn, hck, lck, lyn, src, and yes), all of which diverged from a common evolutionary precursor before the mammalian radiation (25,26,32,34,38,41,43,46,48,49). Each of these genes has the potential to encode a membraneassociated protein tyrosine kinase composed of a specialized amino-terminal domain of about 70 residues that is unique to each src family member joined to a common region containing a prototypical kinase domain. Unlike the related growth factor receptor kinases, which demonstrate ligand-induced increases in kinase activity, physiologic activators of the src-like kinases have not been identified. Thus, the normal function of each of these molecules is obscure. Nevertheless, considerable evidence suggests that the src family kinases can participate in the regulation of cell growth. Three of these kinases (fgr, src, and yes) were originally identified in mutant forms as retroviral oncogenes capable of inducing cell transformation (reviewed in reference 2). Similarly, the Ick gene is rearranged and overexpressed in some murine lymphomas (32,46).Comparison of the deduced structures of the src-like protein tyrosine kinases permits some inferences regarding their physiology. Each of the src family kinases contains an analogous tyrosine residue in a conserved carboxy-terminal sequence context. In pp60c-src this tyrosine (Tyr-527) is phosphorylated in vivo at high stoichiometry (10). Activated forms of pp6Osrc are either hypophosphorylated at Tyr-527, for example, when pp6Oc-src associates with polyomavirus middle T antigen, or lack Tyr-527 altogether, as for pp6v-src (6; reviewed in reference 19). In addition, removal of the phosphate from Tyr-527 in pp60c-rc increases its kinase activity in vitro (12, 13), and mutation of Tyr-527 to phenylalanine increases the k...
The established mouse cell line NIH 3T3 has been used with considerable success over the past three years as the basis of an in vitro transformation assay for demonstrating the presence of transfectable transforming genes in the DNA of certain human and rodent tumour cells (for review see ref. 1). In the case of the human bladder carcinoma cell lines EJ and T24, this approach has led to the molecular cloning of a transforming gene which is closely related to the rat-derived Harvey sarcoma virus oncogene, v-Ha-ras. A single point mutation, which distinguishes these genes from their normal human homologue (c-Ha-ras1), is thought to be solely responsible for their transforming potential. However, carcinogenesis in both humans and laboratory rodents is a multi-stage process (reviewed in ref. 11) of which the NIH 3T3 cell, already partly transformed, may represent only the penultimate stage. We therefore chose to examine the transforming effects of the EJ oncogene in a hamster fibroblast system originally developed in our laboratory to study stages in carcinogen-induced malignant transformation of normal diploid cells. We show here that EJ c-Ha-ras-1 lacks complete transforming activity when transfected into normal fibroblasts which have a limited lifespan, but can fully transform fibroblasts that have been newly 'immortalized' by carcinogens.
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