Water-soluble HPMA copolymer-wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo.

Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)-DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA-DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.

The src homology 2 domain of Bcr/Abl is required for efficient induction of chronic myeloid leukemia-like disease in mice but not for lymphoid leukemogenesis or activation of phosphatidylinositol 3-kinase.

The effect of mutations in the Src homology 2 (SH2) domain of the BCR/ABL oncogene on leukemogenesis was tested in a quantitative murine bone marrow transduction/transplantation assay that accurately models human Philadelphia-positive B-lymphoid leukemia and chronic myeloid leukemia (CML). The SH2 domain was not required for induction of B-lymphoid leukemia in mice by BCR/ABL. Under conditions where the p190 and p210 forms of BCR/ABL induce fatal CML-like myeloproliferative disease within 4 weeks, p210 SH2 mutants induced CML-like disease in some mice only after a significant delay, with other recipients succumbing to B-lymphoid leukemia instead. In contrast, p190 BCR/ABL SH2 point and deletion mutants rapidly induced CML-like disease. These results provide the first direct evidence of significant differences in cell signaling by the Bcr/Abl tyrosine kinase between these distinct leukemias. Contrary to previous observations, high levels of phosphatidylinositol 3-kinase (PI 3-kinase) activity in primary malignant lymphoblasts and myeloid cells from recipients of marrow transduced with the BCR/ABL SH2 mutants were found. Hence, the decreased induction of CML-like disease by the p210 BCR/ABL SH2 mutants is not due to impaired activation of PI 3-kinase.

Inactivation of wild-type BCR/ABL tyrosine kinase in hematopoietic cells by mild hyperthermia.

Temperature-sensitive mutants of BCR/ABL tyrosine kinase have been extensively used to study the mechanisms of cell transformation and signal transduction. However, little is known about the effect of temperature on the activity of wild-type BCR/ABL gene product. In this study, we demonstrate that in vivo tyrosine kinase activity of p210, p190 BCR/ABL and v-abl are temperature-sensitive when expressed in hematopoietic cells and decline when temperature is raised 2 degrees C above normal range. In vitro tyrosine kinase activities of purified recombinant Abl and immunoprecipitated p210 BCR/ABL were also sensitive to increased temperature. Tyrosine phosphorylation of cellular proteins was markedly reduced in BCR/ABL transformed cells after 16 h at 39 degrees C, whereas the expression of BCR/ABL was unchanged. Temperature-induced downregulation of BCR/ABL kinase activity was reversible when cells were shifted back to 37 degrees C. The downregulation of Abl tyrosine kinase activity was not influenced by mutation or deletion of SH2 or SH3 domains or mutation of the GRB2 binding site. No increase in functional activity or expression of protein-tyrosine phosphatases, PTP-1B, SH-PTP1 or SH-PTP2 was detected in cells grown at 39 degrees C. Temperature-induced downregulation in tyrosine kinase activity correlated with decline in phosphotyrosine-associated PI 3-kinase whereas there was no change in growth factor independence of transformed hematopoietic cells. In conclusion, Abl tyrosine kinase has intrinsic sensitivity to temperature and BCR/ABL expressed in hematopoietic cells is downregulated by increasing temperature 2 degrees C. These observations provide a unique opportunity to identify cellular factor(s) which regulate BCR/ABL kinase in vivo and suggests possible novel treatment of CML by a mild hyperthermia.

Generation of antigen-specific cytotoxic T lymphocytes and regulation of cytokine production takes place in the absence of CD3zeta.

The TCR-associated protein CD3zeta plays a major role in regulating the state of responsiveness to peptide-MHC complexes on the surface of antigen-presenting cells. In this paper the requirement of CD3zeta in the generation of cytotoxic T cells was compared with its requirement in cytokine gene activation in two mutant mice: ZKO mice with a disrupted CD3zeta gene and ZTG mice in which a truncated CD3zeta segment was expressed as a transgene on the ZKO background. Upon infection of ZTG mice with lymphocytic choriomeningitis virus (LCMV), antigen-specific cytotoxic T lymphocyte (CTL) responses were detected, identical to responses in wild-type mice. In addition, antigen-specific CTL responses to allogeneic class I and class II MHC in ZTG animals were indistinguishable from those in wild-type animals. However, CTL responses to the same major antigens were not detectable in ZKO mice. We conclude that the signal transduction pathways leading to CTL development and cytokine production can be triggered through TCR in the absence of functional CD3zeta, provided the remainder of the TCR-CD3 complex is expressed at high levels on the cell surface. Surprisingly, IFN-gamma production in response to LCMV followed the same kinetics in ZKO, ZTG and wild-type mice. However, in vitro studies showed that cytokine production in general was abnormally regulated in T lymphocytes from ZKO mice, in contrast to ZTG T cells. Taken together, these studies support the hypothesis that development of CTL can take place in the absence of functional CD3zeta. However, CTL development requires stronger TCR-initiated signal transduction events than induction of cytokine genes.

Tyrosine phosphorylation of the CD3-epsilon subunit of the T cell antigen receptor mediates enhanced association with phosphatidylinositol 3-kinase in Jurkat T cells.

T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3'-kinase (PI 3-kinase). In a previous report, we showed that the p85alpha subunit of the PI 3-kinase preferentially associated with the CD3-zeta membrane-proximal immunoreceptor tyrosine-based activation motif of the zeta chain (zetaA-ITAM) (Exley, M., Varticovski, L., Peter, M., Sancho, J., and Terhorst, C. (1994) J. Biol. Chem. 269, 15140-15146). Here, we demonstrate that tyrosine phosphorylation of CD3-epsilon can recruit the PI 3-kinase enzyme in a T cell activation-dependent manner. In vivo studies with Jurkat cells stably transfected with a CD8-CD3-epsilon chimera (termed CD8-epsilon) shows that ligation of endogenous CD3-epsilon or CD8-epsilon by specific antibodies induces tyrosine phosphorylation of CD3-epsilon or CD8-epsilon, respectively. Increased tyrosine phosphorylation correlates with increased binding of p85alpha PI 3-kinase and recruitment of PI 3-kinase enzymatic activity to CD3-epsilon or CD8-epsilon proteins. Mutagenesis studies in COS-7 cells, transiently transfected with CD8-epsilon, p85alpha, and Fyn cDNAs in various combinations, show that both Tyr170 and Tyr181 within the CD3-epsilon-ITAM are required for efficient binding of p85alpha PI 3-kinase. Thus, replacement of Tyr170 by Phe (Y170F), or Tyr181 by Phe (Y181F) significantly reduces binding of p85alpha PI 3-kinase, whereas it does not affect binding of Fyn. Further in vitro experiments suggest that a direct binding of the tandem SH2 domains of p85alpha PI 3-kinase to the two phosphorylated tyrosines in a single CD3-epsilon-ITAM may occur. The data also support a model in which a single CD3 subunit can recruit distinct effector molecules by means of TCR-mediated differential ITAM phosphorylation.