TSA Cells Research Articles

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.

35 Preclinical model for gene therapy: Role of the host

Three different experimental systems based on cytokine gene transduction can provide evidence that (I) systemic immunity does not always follow tumor regression; (II) a cytokine combination that efficiently induces systemic immunity does not induce CTL activity and does not exert therapeutic effects; (III) a different cytokine combination induces both CTL and protection in Winn assay with out inhibiting tumor take and outgrowth. (I) C-26/G-CSF regression is coupled with infiltration of leukocytes releasing secondary cytokines and depends on CD8 + T cells, regressor mice however, remain susceptible to a challenge with C-26 cells. (II) TSA/IL-4 more efficiently than TSA/IL-2 induce protection against a challenge with live TSA cells. To transfer IL-4 mediated systemic immunity, both lymphocytes and serum from immune mice are needed. TSA/IL-4 cells when used as vaccine to cure TSA bearing mice were with out effect, where as TSA/IL-2 were moderately effective. (III) C-26/IL-12 cells showed delayed tumor onset that was NK dependent. Immunocytochemical characterisation of leukocytes infiltrating C-26/IL-12 tumors showed few infiltrating T cells in non-depleted mice but abundant infiltration by CD8 + T cells in tumors from mice depleted ofCD4 + T cells and CD4 depletion allowed tumor regression in about 30% of mice. This is not due to a CD4-mediated suppression since mice primed with C-26/IL-12 cells possessed lytic lymphocytes and CD8 + T cell which mediated protection against C-26/IL-12 in a Winn assay.

Differential regulation of cAMP by endogenous versus transfected fornylpeptide chemoattractant receptors: Implications for G i-coupled receptor signaling

Endogenous neutrophil formylpeptide receptors do not inhibit adenylylcyclase activation. The ability of a cloned and transfected human formylpeptide receptor to mediate the inhibition of adenylylcyclase was assessed in the human embryonic kidney 293 TSA cell line. Inclusion of 1 microM fMetLeuPhe resulted in a ca. 50% inhibition of isoproterenol-stimulated cAMP in transfected cells. Activation of adenylylcyclase by isoproterenol was inhibited ca. 30% by fMetLeuPhe in membranes prepared from transfected cells but not in membranes prepared from neutrophils. Prior treatment of transfected cells with pertussis toxin abrogated the inhibitory effect of fMetLeuPhe. These data indicate that factors in addition to the primary structure of the formylpeptide receptor govern its transductional activities.

Functional high efficiency expression of cloned leucocyte chemoattractant receptor cDNAs

Human kidney 293 TSA cells were transfected by a calcium phosphate method with human formylpeptide and C5a receptor cDNAs with high efficiency. Formylpeptide receptor positive transfectants expressed a total of 968,000 ± 34,000 receptors per cell with two affinity states ( K ds of ca. 0.43 nM and 39 nM), which in the presence of 100μM GTPγS decreased by ca. 4-fold the number of high-affinity sites. The ligand binding pharmacology of cloned and expressed formylpeptide receptors were indistinguishable from endogenous receptors on human neutrophils. Expressed formylpeptide and C5a receptors were functionally active in mobilizing intracellular calcium via a pertussis toxin sensitive mechanism with an ED 50 for formylpeptide of ca. 0.5–1.0 nM. This expression system, in which receptor expression can be monitored by flow cytometric methods and in which intracellular calcium responses are measurable, unlike in the more popular COS-7 cell expression system, will provide a useful basis for the analysis of chemoattractant receptor structure-function relationships.

Immunoregulation of lysozyme-specific suppression III. Epitope-specific amplification of immunosuppression induced by monoclonal suppressor-T-cell products

The hen egg-white lysozyme (HEL)-specific suppression induced by soluble molecules produced by a monoclonal T-cell lymphoma line (LH8-105) obtained from HEL-specific suppressor T lymphocytes has been examined. Injection of I-J+ molecules from LH8-105 cell culture supernatant (TsFa) in HEL-primed mice during the afferent phase of the response induced Lyt-2+ second order suppressor T (Ts) cells which, upon transfer into HEL-CFA-primed syngeneic recipients, inhibit the delayed-type hypersensitivity (DTH) response to HEL. Transfer of spleen cells from TsFa-injected mice primed with HEL or human lysozyme suppresses the DTH response to HEL in recipient mice whereas this response is not affected by cell transfer from ring-necked pheasant egg-white lysozyme (REL)-primed and TsFa-injected mice, indicating that induction of second order Ts by TsFa is specific for a lysozyme epitope including phenylalanine at position 3. Fine antigenic specificity of second order Ts-cell induction is confirmed by similar results obtained upon injection of TsFa in mice primed with HEL N-terminal synthetic peptide or with an analog in which, as in REL, phenylalanine has been substituted by tyrosine at position 3. The same fine antigenic specificity observed in the induction of second order Ts cells is also present in the expression of TsFe suppressive activity. The similar antigenic specificity of Tsa and Tse suggests that Tse cells could result from amplification of the Tsa cell population or these two cell subsets could reflect different maturation stages of the same cell type rather than distinct T-cell populations activated in cascade.

Tumorigenicity of tsA and wild‐type simian virus 40 transformed cells inoculated onto the chicken chorioallantoic membrane

This study compares the tumorigenicity of SV40 primary tumor cell lines, tsA and wild-type SV40-transformed Chinese hamster cells, at two temperatures, 37 degrees C and 40.5 degrees C, inoculated onto the chorioallantoic membrane of the chicken egg. The SV40 primary tumor cell lines varied in their efficiency of takes at 37 degrees C from 78% for the H65-90B tumor line, 73% for the H80-7A and 25% for the H80-4 line. At 40.5 degrees C the H80-4 was unable to form tumors; however, the H80-7A and H80-4 produced 70% and 20% tumors respectively. Histologically, all CAM tumors were fibrosarcomas identical to the transplanted tumors, however, the tumor(s) at 40.5 degrees C were smaller. Chinese hamster wild-type SV40-transformed cells were equally efficient (32%) at tumor production at both temperatures. The tsA-SV40-transformed Chinese hamster cells (A58 and A58-2) induced 34% tumors at 37 degrees C and 9% tumors at 40.5 degrees C. At 37 degrees C these tumors were typical fibrosarcomas; however, the 40.5 degrees C tumors were smaller and less cellular, resembling a more differentiated fibrosarcoma. Therefore, the tsA Chinese hamster transformed cells were less efficient at tumor induction at the non-permissive temperature; however, the primary tumor lines also demonstrated a variability in tumorigenicity (H65-90B and H80-4). Possibly factors other than the temperature-sensitive viral protein (big "T" antigen) may be involved in establishing a tumor on the chicken CAM.

The requirement of simian virus 40 gene A product for the stimulation of cellular thymidine kinase activity after viral infection

Mouse cells in culture contain two distinct forms of thymidine kinase enzyme activities. These two enzymes have been separated by polyacrylamide gel electrophoresis into a 0.2 R f and a 0.5 R f activity. The 0.2 R f enzyme was found only in actively growing cells, while the 0.5 R f form of thymidine kinase is the mitochondrial-associated enzyme and is most prominent in resting cells in culture. SV40 infection of these resting cells results in an increased specific activity of only the 0.2 R f form of this enzyme. SV40 wild-type and SV40 tsBC and tsC mutants stimulated the levels of the 0.2 R f thymidine kinase in resting cells after viral infection at either the permissive temperature (32°) or the nonpermissive temperature (41°). Five different SV40 tsA mutants ( tsA7, 28, 30, 58, and 209) and two different SV40 tsD mutants ( tsD202, 270) only stimulated thymidine kinase activity at the permissive temperature. Little or no 0.2 R f thymidine kinase activity could be detected in tsA or tsD mutant-infected cells at the nonpermissive temperature. The SV40 tsA255 mutant appeared to be an exception to the A mutant class in that it stimulated the 0.2 R f thymidine kinase activity at both permissive and nonpermissive temperatures. These results indicate that the SV40 A gene product may be required directly, and the D gene product indirectly, in the stimulation of cellular enzyme activities following viral infection.