In vitro microtumors provide a physiologically predictive tool for breast cancer therapeutic screening.

Many anti-cancer drugs fail in human trials despite showing efficacy in preclinical models. It is clear that the in vitro assays involving 2D monoculture do not reflect the complex extracellular matrix, chemical, and cellular microenvironment of the tumor tissue, and this may explain the failure of 2D models to predict clinical efficacy. We first optimized an in vitro microtumor model using a tumor-aligned ECM, a tumor-aligned medium, MCF-7 and MDA-MB-231 breast cancer spheroids, human umbilical vein endothelial cells, and human stromal cells to recapitulate the tissue architecture, chemical environment, and cellular organization of a growing and invading tumor. We assayed the microtumor for cell proliferation and invasion in a tumor-aligned extracellular matrix, exhibiting collagen deposition, acidity, glucose deprivation, and hypoxia. We found maximal proliferation and invasion when the multicellular spheroids were cultured in a tumor-aligned medium, having low pH and low glucose, with 10% fetal bovine serum under hypoxic conditions. In a 7-day assay, varying doses of fluorouracil or paclitaxel had differential effects on proliferation for MCF-7 and MDA-MB-231 tumor spheroids in microtumor compared to 2D and 3D monoculture. The microtumors exhibited a tumor morphology and drug response similar to published xenograft data, thus demonstrating a more physiologically predictive in vitro model.

Lack of compartmentalization of HIV-1 quasispecies between the gut and peripheral blood compartments.

Compartmental differences in human immunodeficiency virus type 1 (HIV-1) between the gut and peripheral blood and within the gut were examined. Biopsy specimens from the colon and ileum and peripheral blood samples were collected from chronically HIV-1-infected individuals. HIV-1 envelope sequences were examined from cell-associated DNA and RNA and virion RNA. Phylogenetic analysis revealed no evidence of compartmentalization of HIV-1 between the gut and peripheral blood and within the gut (colon and ileum). HIV-1 sequences detected in the gut were transcriptionally active and were also found in peripheral blood from matching time points, providing evidence of ongoing virus production in the gut and equilibrium of HIV-1 between the gut and peripheral blood compartments.

HIV-1 viruses detected during episodic blips following interleukin-7 administration are similar to the viruses present before and after interleukin-7 therapy.

BACKGROUND: administration of recombinant human interleukin (IL)-7 leads to CD4 and CD8 T-cell expansions in HIV-infected individuals, demonstrating promising capacity for immune reconstitution. However, a proportion of patients treated with recombinant human IL-7 experience transient increases in plasma HIV-RNA ('blips'), possibly reflecting 'purging' of a quiescent reservoir that provides a barrier to viral eradication. OBJECTIVE: to identify the sources of HIV detected during transient viremic episodes following IL-7 administration, viral quasispecies were analyzed in a total of 281 primary sequences derived from seven patients who experienced the episodic blips following IL-7 therapy. METHOD: the C2-V3 regions of the HIV-1 env gene were sequenced from HIV-1 RNA in plasma and HIV DNA from peripheral blood mononuclear cells (PBMCs) obtained at baseline (day 0 of recombinant human IL-7 therapy), during the episode of viral blips (day 4), and at a time when levels of plasma HIV-RNA had returned to less than 50 copies/ml (day 28). RESULTS: the HIV sequences detected during transient viremia following IL-7 administration were closely related to those of the plasma viruses present before and after cytokine administration. All virus quasispecies detected during blips were also present in proviral sequences in PBMCs. CONCLUSION: the low level viremia induced by IL-7 likely reflects predominantly transient induction or release of virus from a preexisting pool rather than activation of silent quasispecies.

Zap70 signaling pathway mediates glucocorticoid receptor-dependent transcriptional activation: role in the regulation of annexin 1 expression in T cells.

We have recently shown that Zap70 is important in retinoid receptor-dependent transactivation in T lymphocytes. We report that Zap70 signaling is also essential in dexamethasone-inducible glucocorticoid receptor (GR)-mediated transactivation in T lymphocytes. Zap70-negative Jurkat T cells and cells reconstituted with inactive Zap70 exhibited attenuated GR-mediated activation as compared with Zap70 reconstituted and wild-type cells. Lck-lacking Jurkat cells were also found to show markedly reduced GR activation, and reconstitution with Lck restored the activation. Gene array and protein analysis showed that the level of annexin 1 (ANXA1), an anti-inflammatory protein known to be induced and released by the glucocorticoid action, was significantly reduced in Zap70-negative and Zap70-inactive Jurkat cells as compared with wild-type cells. Lck-lacking cells were also found to have markedly reduced ANXA1 levels and reconstitution with Lck restored the ANXA1 expression. RNA interference-induced knockdown of Zap70 or Lck in Jurkat cells and peripheral blood T lymphocytes also resulted in the loss of ANXA1 expression. Transcriptional analysis revealed that dexamethasone-inducible GR-mediated activation of ANXA1 promoter was compromised in both Zap70 knocked down peripheral blood T cells and Zap70 or Lck-deficient/Lck-inactive Jurkat cells, indicating an essential role of these kinases in GR-mediated ANXA1 promoter activation in T lymphocytes. To summarize, our data demonstrate an important role for Zap70 signaling in GR-mediated transactivation in T lymphocytes and also point out a crucial role of this kinase in maintaining normal ANXA1 levels in these cells.

Evidence for the involvement of tyrosine kinase ZAP 70 in nuclear retinoid receptor-dependent transactivation in T lymphocytes.

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are transcription factors that control diverse cellular functions during development and homeostasis. The biochemical role of these proteins in T lymphocytes is not well known. Here we have studied the role of protein-tyrosine kinase ZAP 70, a key enzyme involved in the proximal signaling events during T cell activation, in the modulation of RXRE- and RARE-dependent activation in T lymphocytes. Surprisingly, ZAP 70-negative Jurkat T cells showed considerable loss of both RXRE- and RARE-mediated transactivation as compared with wild type Jurkat cells. In addition, ZAP 70-negative cells failed to exhibit normal protein kinase C and calcineurin-induced transcriptional activity. ZAP 70-negative cells that were reconstituted with active ZAP 70 regained the transactivation function, whereas cells expressing kinase-dead form of ZAP 70 failed to do so. Defective transcriptional activation was also observed in actively proliferating human peripheral blood T lymphocytes in which RNA interference was used to induce loss of ZAP 70 expression. In addition, an Lck-deficient Jurkat cell line that cannot efficiently activate ZAP 70 was also found defective in RXRE-mediated transcription. Finally, RNA interference-induced loss of ZAP 70 or Lck protein in Jurkat cells resulted in significant decrease in the RXRE-dependent activation. Together, these results suggest a novel functional role for ZAP 70 in nuclear receptor-driven transactivation in T lymphocytes.

Protein kinase C theta modulates nuclear receptor-corepressor interaction during T cell activation.

Transcriptional repression by nuclear receptor corepressors plays a critical role in T cell development. However, the role of these corepressors in T cell activation is poorly understood. We report that T cell activation silenced transcription driven by nuclear receptors retinoic acid receptor, retinoid X receptor, and thyroid hormone receptor and induced silencing mediator of retinoic acid and thyroid hormone receptors (SMRT)-receptor interaction. Whereas the expression of a dominant active mutant of protein kinase C theta(PKC theta) induced strong SMRT-receptor interaction in the absence of T cell activation, a dominant negative mutant of PKC theta decreased the interaction. Loss of PKC theta expression by induction of "RNA interference" resulted in the attenuation of basal and activation-induced SMRT-receptor interaction. We suggest that T cell activation silences nuclear receptor-dependent transactivation in part through PKC theta-dependent enhancement of SMRT-receptor interaction.