A role for GATA transcription factors in the androgen regulation of the prostate-specific antigen gene enhancer

The transgenic mouse line Gγ/T-15 containing the fetal globin promoter linked to SV40 T antigen unexpectedly results in androgen-independent prostate carcinomas. Given the key role of GATA-1 transcription factor in fetal globin gene promoter activity, we investigated whether specific GATA family members are expressed in the prostate and whether they can regulate prostate-specific genes. We found that GATA-2 and -3 are the predominant GATA family members expressed in human and mouse prostate and that GATA mRNA levels are not regulated by androgen. We identified six GATA sites flanking an androgen-response element located in the far-upstream enhancer of the prostate-specific antigen (PSA) gene. These GATA sites are targets for GATA factors and are essential for optimal androgen induction of transfected PSA enhancer/promoter plasmids in LNCaP, a PSA and androgen receptor expressing human prostate cancer cell line. Our results suggest that prostatic GATA-2 and -3 are involved in the androgen regulation of the PSA gene

Extracts from two marine sponges lower cyclin B1 levels, cause a G2/M cell cycle block and trigger apoptosis in SW-13 human adrenal carcinoma cells

Marine sponges have been shown to produce metabolites with cell growth- and endocrine-altering activities. We tested extracts from two species: the ‘brown variable sponge’ ( Anthosigmella varians) and the ‘West Indian bath sponge’ ( Spongia barbara), for effects on the cell cycle regulatory protein, cyclin B1; cell cycle growth-phase (sub-G1/apoptosis, G1, S, and G2/M); and cell survival in SW-13 human adrenal carcinoma cultures. Polyacrylamide gel electrophoresis studies indicated a 70–90% reduction in cyclin B1 levels by treatment with these agents. Microscopic examination of cultures with DAPI staining showed dense and fragmented DNA fluorescence, characteristic of apoptosis, in both sponge extract-treated cultures but not in controls. Flow cytometry analysis showed a 16-fold increase in the percentage of cells entering apoptosis (sub-G1 phase of cell cycle) by treatment with Anthosigmella varians extract ( p<0.01) and a 10-fold increase using Spongia barbara extract ( p<0.01). During this same time, the percentage of cells in G2/M was increased 1.6-fold by Anthosigmella varians extract ( p<0.01) and 2.0-fold by Spongia barbara extract ( p<0.01). Cell growth/survival studies also indicated a time-dependent decline in the percentage confluence of cell cultures exposed to Anthosigmella varians or Spongia barbara extracts. These experiments demonstrate that some species of marine sponges have metabolites which are capable of interfering with the mammalian cell cycle and with the survival of human adrenal carcinoma cells in culture

2-Methoxyestradiol Induces G2/M Arrest and Apoptosis in Prostate Cancer

Few therapeutic treatment options are available for patientssuffering from metastatic androgen-independent prostate cancer. We investigated the ability of the estrogen metabolite 2-methoxyestradiol to inhibit the proliferation of a variety of human prostate cancer cell lines in vitro and to inhibit the growth of androgen-independent prostate cancer in a transgenic mouse model in vivo. Our results showed that 2-methoxyestradiol is a powerful growth inhibitor of LNCaP, DU 145, PC-3, and ALVA-31 prostate cancer cells. Cell flow cytometry of 2-methoxyestradiol-treated DU 145 cells showed a marked accumulation of cells in the G2/M phase of the cell cycle and an increase in the sub-G1 fraction (apoptotic). In addition, staining for annexin V, changes in nuclear morphology, and inhibition of caspase activity support a role for apoptosis. More importantly, we showed that 2-methoxyestradiol inhibits prostate tumor progression in the Gγ/T-15 transgenic mouse model of androgen-independent prostate cancer without toxic side effects. These results in cell culture and an animal model support investigations into the clinical use of 2-methoxyestradiolin patients with androgen-independent prostate cancer

Human marrow-isolated adult multilineage-inducible (MIAMI) cells protect against peripheral vascular ischemia in a mouse model

The treatment of peripheral vascular disease (PVD) with stem cells potentially offers a promising strategy. We tested marrow-isolated adult multilineage-inducible (MIAMI) cells to induce neovascularization in a mouse model of critical hindlimb ischemia (CLI). CLI was induced in the right hindlimb of Balb/C mice. One million MIAMI cells, normally grown at 3% O2, were injected in the adductor muscle along the ischemic region. All animals (n=11 per group) were immunosuppressed with cyclosporine daily for the entire period. Human foreskin fibroblast (HFF) cells and phosphate-buffered saline (PBS) were used as controls. Blood perfusion in the ischemic right and non-ischemic left hindlimbs was measured. Compared with animals receiving HFF cells or PBS, MIAMI cells significantly improved blood perfusion, necrosis and inflammation in the ischemic limb. A fraction of injected MIAMI cells expressed CD31 and von Willebrand factor (vWF). MIAMI cells in vitro, under pro-angiogenic growth conditions, differentiated into endothelial-like cells and expressed endothelial markers such as CD31 and vWF, determined by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR), and CD31 and kinase insert domain receptor (KDR), determined by immunofluorescence. Moreover, MIAMI cells formed vascular endothelial-like tubules in the presence of matrigel. Bioplex immunoassay analysis showed increased secretion of angiogenic/anti-inflammatory factors by the MIAMI cells under 3% O2 compared with 21% O2, including monocyte chemoattractant protein-1 (MCP-1), fractalkine (Ftk), growth-related oncogene (GRO), vascular endothelial growth factor (VEGF), interleukin (IL)-6 and IL-8. Furthermore, transcripts for anti-inflammatory molecules stanniocalcin-1 (STC-1) and tumor necrosis factor-α-stimulated gene 6 (TSG-6) were up-regulated several fold. MIAMI cells can be very useful for patients affected by CLI. MIAMI cells promote blood vessel formation and reduce inflammation and necrosis in ischemic tissue

MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways

Oncogenic RAS promotes production of reactive oxygen species (ROS), which mediate pro-malignant signaling but can also trigger DNA damage-induced tumor suppression. Thus RAS-driven tumor cells require redox-protective mechanisms to mitigate the damaging aspects of ROS. Here we show that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase that sanitizes oxidative damage in the nucleotide pool, is important for maintaining several KRAS-driven pro-malignant traits in a nonsmall cell lung carcinoma (NSCLC) model. MTH1 suppression in KRAS-mutant NSCLC cells impairs proliferation and xenograft tumor formation. Furthermore, MTH1 levels modulate KRAS-induced transformation of immortalized lung epithelial cells. MTH1 expression is upregulated by oncogenic KRAS and correlates positively with high KRAS levels in NSCLC human tumors. At a molecular level, in p53-competent KRAS-mutant cells, MTH1 loss provokes DNA damage and induction of oncogene-induced senescence (OIS). In p53-nonfunctional KRAS-mutant cells, MTH1 suppression does not produce DNA damage but induces a reduced proliferative rate and an adaptive decrease in KRAS expression levels. Thus, MTH1 not only enables evasion of oxidative DNA damage and its consequences but can also function as a molecular rheostat for maintaining oncogene expression at optimal levels. Accordingly, our results indicate MTH1 is a novel and critical component of oncogenic KRAS-associated malignancy and its inhibition is likely to yield significant tumor-suppressive outcomes in KRAS-driven tumors

Low Oxygen Modulates Multiple Signaling Pathways, Increasing Self-Renewal, While Decreasing Differentiation, Senescence, and Apoptosis in Stromal MIAMI Cells

Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and β-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells