High Cks1 expression in transgenic and carcinogen-initiated mammary tumors is not always accompanied by reduction in p27Kip1.

Cks1 plays an essential role in SCFSkp2-mediated ubiquitination, and consequently turnover, of the cdk2 inhibitor and tumor supressor p27Kip1. High Cks1 expression is associated with aggressive breast tumors and correlates with low p27Kip1 levels in some cases, although it is also an independent prognostic marker for survival, and provides predictive information in addition to that provided by p27Kip1 alone. In this report we demonstrate that Cks1 protein and mRNA are elevated to very high levels in mammary tumors initiated by erbB2, c-myc and polyoma middle-T (PyMT) in transgenic mice, whereas Cks1 protein is hardly detectable in the normal mammary epithelium. Cks1 is also highly upregulated in rat mammary tumors initiated by methylnitrosourea (MNU). Despite high levels of Cks1 expression, p27Kip1 levels were not reduced, and were in fact slightly higher in mammary tumors initiated by erbB2, PyMT and MNU. In contrast mammary tumors from MMTV-c-myc mice did exhibit low p27Kip1 and higher levels of Skp2. Together, these data suggest that deregulated Cks1 expression might play roles in oncogene and carcinogen-initiated mammary tumorigenesis independent of p27Kip1 turnover in certain tumors. Stable overexpression of Cks1 in human breast carcinoma MCF-7 cells did not significantly reduce p27Kip1 expression, although it conferred resistance to Faslodex (ICI 182780)-mediated inhibition of colony outgrowth in these cells. In contrast, Cks1-depleted MCF-7 cells formed fewer colonies in estrogen-containing medium. Therefore, our studies also suggest that Cks1 levels regulate the responsiveness of ER+ breast cancers to estrogens and anti-estrogens.

Vascular endothelial growth factor reduces tamoxifen efficacy and promotes metastatic colonization and desmoplasia in breast tumors.

Clinical studies have shown that decreased tamoxifen effectiveness correlates with elevated levels of vascular endothelial growth factor (VEGF)-A(165) in biopsy samples of breast cancers. To investigate the mechanisms underlying tamoxifen resistance and metastasis, we engineered the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line to express VEGF to clinically relevant levels in a doxycycline-regulated manner. Induction of VEGF expression in orthotopically implanted xenografts that were initially tamoxifen responsive and noninvasive resulted in tamoxifen-resistant tumor growth and metastasis to the lungs. Lung metastases were also observed in a VEGF-dependent manner following tail vein injection of tumor cells. At both primary and metastatic sites, VEGF-overexpressing tumors exhibited extensive fibroblastic stromal content, a clinical feature called desmoplasia. VEGF-induced metastatic colonies were surrounded by densely packed stromal cells before detectable angiogenesis, suggesting that VEGF is involved in the initiation of desmoplasia. Because expression of VEGF receptors R1 and R2 was undetectable in these tumor cells, the observed VEGF effects on reduction of tamoxifen efficacy and metastatic colonization are most likely mediated by paracrine signaling that enhances tumor/stromal cell interactions and increases the level of desmoplasia. This study reveals new roles for VEGF in breast cancer progression and suggests that combination of antiestrogens and VEGF inhibitors may prolong tamoxifen sensitivity and prevent metastasis in patients with ER-positive tumors.

Cks1 regulates cdk1 expression: a novel role during mitotic entry in breast cancer cells.

Cks1, a small protein whose expression is strongly associated with aggressive breast tumors, is a component of cyclin-cdk complexes, as well as the SCF(Skp2) ubiquitin ligase. In these studies, we explored its roles in estrogen receptor-positive breast tumor cells. When exposed to the antiestrogen ICI 182780, these cells accumulate in G(1) by reducing the expression of Cks1, and increasing the levels of p130/Rb2, a cdk2 inhibitor and SCF(Skp2) target. Heregulin beta1 or estradiol abrogate antiestrogen effects by increasing Cks1 expression, down-regulating p130/Rb2 and inducing S phase entry. Depletion of Cks1 in these cells by RNA interference concomitantly decreased Skp2 and up-regulated p130/Rb2 and another SCF(Skp2) target, p27(Kip1). Remarkably, however, Cks1-depleted cells not only exhibit slowed G(1) progression, but also accumulate in G(2)-M due to blocked mitotic entry. Notably, we show that cdk1 expression, which is crucial for M phase entry, is drastically diminished by Cks1 depletion, and that restoration of cdk1 reduces G(2)-M accumulation in Cks1-depleted cells. cdk1 reduction in Cks1-depleted cells is a consequence of a marked decrease in its mRNA and not due to alteration in its proteolytic turnover. Both heregulin beta1 and estradiol could neither restore cdk1 nor sustain cycling in Cks1-depleted cells, although classical estrogen receptor function remained unaltered. Cks1 depletion also decreased Skp2 in human mammary epithelial cells without altering cell cycle progression. Thus, the indispensability of Cks1 to the breast cancer cell cycle, versus its redundancy in normal cells, suggests that Cks1 abrogation could be an effective interventional strategy in breast cancer.

Efficacy of rifabutin-loaded microspheres for treatment of Mycobacterium avium-infected macrophages and mice.

Two poly(DL-lactide-co-glycolide) microsphere formulations (A, 10% wt/wt, and B, 23% wt/wt, 1-10 microns) were evaluated for intracellular delivery of rifabutin using the J774 murine and Mono Mac 6 (MM6) human monocytic cell lines. Within 7 days, formulation A released 100% in both cell lines and B released 53 and 67% in the J774 and MM6, respectively. Intracellular release of rifabutin with both formulations caused significant reduction of intracellularly replicating Mycobacterium avium (MAC). In MAC-infected beige mice, formulation B (50 mg, intraperitoneal days 0 and 7) completely eliminated infection by 21 days (p < 0.001), similar to a rifabutin daily oral regimen.

MEK ablation in MCF-7 cells blocks DNA synthesis induced by serum, but not by estradiol or growth factors.

The role of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling in estrogen receptor positive (ER(+)) MCF-7 breast carcinoma cells is not well understood. We depleted MEK by cotransfection of MEK1 and MEK2 siRNA duplexes in a MCF-7 derived line (MCF-7/ lacZ, ML-20) and determined its effect on serum, 17beta-estradiol (E(2)), and growth factor induced DNA synthesis. MEK knockdown did not decrease fetal bovine serum-induced DNA synthesis in ML-20 cells although it did inhibit DNA synthesis induced by estrogen-stripped calf serum (CCS) suggesting that MEK activation plays an important role in growth signaling induced by serum components other than estrogen. Consistent with this notion, MEK knockdown only modestly decreased DNA synthesis induced by E(2)-supplemented CCS medium in ML-20 cells. Similarly, MEK knockdown only caused moderate decreases in DNA synthesis induced by fibroblast growth factor-1 (FGF-1) or heregulin-beta1 (HRGbeta1) in this media. Also, there were only minimal effects of MEK knockdown in cells treated with growth factor-supplemented serum-free medium. Although MEK depletion inhibited ERK1/2 phosphorylation induced by CCS in these cells, that induced by growth factor supplemented CCS media was relatively unaffected. Similarly, ERK1/2 phosphorylation induced by growth factor-supplemented serum-free media was also relatively unaffected by MEK depletion. These results suggest that pathways regulating DNA synthesis induced by serum in MCF-7 cells are significantly more dependent on constitutive MEK levels than that induced by E(2) or growth factors.

c-Abl-independent p73 stabilization during gemcitabine- or 4'-thio-beta-D-arabinofuranosylcytosine-induced apoptosis in wild-type and p53-null colorectal cancer cells.

Nucleoside anticancer drugs like gemcitabine (2'-deoxy-2',2'-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4'-thio-beta-d-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53-/- HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53-/- and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis.

Prolonged extracellular signal-regulated kinase 1/2 activation during fibroblast growth factor 1- or heregulin beta1-induced antiestrogen-resistant growth of breast cancer cells is resistant to mitogen-activated protein/extracellular regulated kinase kinase inhibitors.

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin beta1 (HRGbeta1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGbeta1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGbeta1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGbeta1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGbeta1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGbeta1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.

Homogeneity and long-term stability of tetracycline-regulated gene expression with low basal activity by using the rtTA2S-M2 transactivator and insulator-flanked reporter vectors.

Inducible expression of tetracycline responsive element (TRE)-regulated genes in nearly all cells in a stable clone has generally been problematic, especially in long-term culture. Heterogeneity of tet-inducible expression is generally attributed to the instability of the original tet-transactivators tTA and rtTA. These transactivators have cryptic splice sites, prokaryotic codons and full VP16 domains, all of which contribute to their instability. Moreover, they also require high concentrations of Doxycycline (Dox). The 5 amino acid substitutions in the rtTA variant rtTA2S-M2 confer exquisite sensitivity to Dox. Moreover, humanized codons, removal of cryptic splice sites and minimal VP16 domains in rtTA2S-M2 results in its being better tolerated within cells. However, the ability of this modified transactivator to maintain homogeneous inducibility in long-term culture has not been examined. We demonstrate that rtTA2S-M2 expressing clones exhibit functional transactivator activity for over 7 months in culture. Furthermore, rtTA2S-M2 expressing clones with chromosomally integrated copies of a TRE-green fluorescent protein (GFP) reporter also exhibited homogeneous inducibility in long-term culture. Importantly, the inherent reduced toxicity and improved stability of rtTA2S-M2 obviates the need to continuously select for its message, once clones with functional transactivator are isolated. The use of rtTA2S-M2 did not, however, preclude clones with stably integrated TRE-reporter from exhibiting leakiness. However, inclusion of flanking double copies of a 'minimal core element' of the chicken beta-globin gene insulator, instead of the 1.4 kb region, in the TRE-reporter was sufficient to markedly reduce the frequency of clones with high basal expression. Inclusion of the insulator core also did not affect the maximal expression levels of the inducible gene, which typically equaled or exceeded that observed with the strong constitutive CMV promoter. Finally, with this system homogeneous inducibility was observed rapidly and with low doses of Dox.

Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis.

Naturally occurring anti-HIV-1 agent (+)-calanolide A was found to be active against all of the strains of Mycobacterium tuberculosis tested, including those resistant to the standard antitubercular drugs. Efficacy evaluations in macrophages revealed that (+)-calanolide A significantly inhibited intracellular replication of M. tuberculosis H37Rv at concentrations below the MIC observed in vitro. Preliminary mechanistic studies indicated that (+)-calanolide A rapidly inhibits RNA and DNA synthesis followed by an inhibition of protein synthesis. Compared with known inhibitors, this scenario is more similar to effects observed with rifampin, an inhibitor of RNA synthesis. Since (+)-calanolide A was active against a rifampin-resistant strain, it is believed that these two agents may involve different targets. (+)-Calanolide A and its related pyranocoumarins are the first class of compounds identified to possess antimycobacterial and antiretroviral activities, representing a new pharmacophore for anti-TB activity.

Antimycobacterial activity of 2-methyl-adenosine.

OBJECTIVES: The aims of this study were to assess the in vitro activity of 2-methyl-adenosine against Mycobacterium tuberculosis and evaluate, and to intracellular efficacy, and to evaluate its effectiveness against M. tuberculosis in a persistent state model and examine its potential mechanism of action. METHODS: In vitro activity was determined by means of a colorimetric microdilution broth assay. Intracellular activity was assessed with a Mono Mac 6 human monocytic cell line. A hypoxic shift-down model was used to evaluate the effect of 2-methyl-adenosine on M. tuberculosis in a persistent state. Mechanism-of-action studies were conducted by examining the effect of 2-methyl-adenosine on the uptake of appropriate radiolabelled precursors into respective mycobacterial macromolecular components. RESULTS: Studies confirmed the in vitro activity of 2-methyl-adenosine against M. tuberculosis and demonstrated intracellular efficacy against M. tuberculosis within macrophages. 2-Methyl-adenosine was able to significantly affect the viability of M. tuberculosis in a hypoxic shift-down model previously described to simulate the persistent state that results during tuberculosis. Mechanism-of-action studies revealed that the immediate inhibitory effects of 2-methyl-adenosine were associated with protein and DNA synthesis and not RNA synthesis. CONCLUSIONS: Results indicate that 2-methyl-adenosine, or similar derivatives, might be effective against M. tuberculosis infections during latency. This information should be helpful in understanding purine metabolism of M. tuberculosis and also the metabolic activity of this important human pathogen in the persistent state.