Dictyostelium discoideum cells secrete CfaD, a protein that is similar to cathepsin proteases. Cells that lack cfaD proliferate faster and reach a higher stationary-phase density than wild-type cells, whereas cells that overexpress CfaD proliferate slowly and reach the stationary phase when at a low density. On a per-nucleus basis, CfaD affects proliferation but not growth. The drawback of not having CfaD is a reduced spore viability. Recombinant CfaD has no detectable protease activity but, when added to cells, inhibits the proliferation of wild-type and cfaD– cells. The secreted protein AprA also inhibits proliferation. AprA is necessary for the effect of CfaD on proliferation. Molecular-sieve chromatography indicates that in conditioned growth medium, the 60 kDa CfaD is part of a ∼150 kDa complex, and both chromatography and pull-down assays suggest that CfaD interacts with AprA. These results suggest that two interacting proteins may function together as a chalone signal in a negative feedback loop that slows Dictyostelium cell proliferation.

Tuberin, the Tsc2 gene product, integrates the phosphatidylinositol 3-kinase/mitogen-activated protein kinase (mitogenic) and LKB1/AMP-activated protein kinase (AMPK; energy) signaling pathways, and previous independent studies have shown that loss of tuberin is associated with elevated AMPK signaling and altered p27 function. In Tsc2-null tumors and tumor-derived cells from Eker rats, we observed elevated AMPK signaling and concordant cytoplasmic mislocalization of p27. Cytoplasmic localization of p27 in Tsc2-null cells was reversible pharmacologically using inhibitors of the LKB1/ AMPK pathway, and localization of p27 to the cytoplasm could be induced directly by activating AMPK physiologically (glucose deprivation) or genetically (constitutively active AMPK) in Tsc2-proficient cells. Furthermore, AMPK phosphorylated p27 in vitro on at least three sites including T170 near the nuclear localization signal, and T170 was shown to determine p27 localization in response to AMPK signaling. p27 functions in the nucleus to suppress cyclin-dependent kinase-2 (Cdk2) activity and has been reported to mediate an antiapoptotic function when localized to the cytoplasm. We found that cells with elevated AMPK signaling and cytoplasmic p27 localization exhibited elevated Cdk2 activity, which could be suppressed by inhibiting AMPK signaling. In addition, cells with elevated AMPK signaling and cytoplasmic p27 localization were resistant to apoptosis, which could be overcome by inhibition of AMPK signaling and relocalization of p27 to the nucleus. These data show that AMPK signaling determines the subcellular localization of p27, and identifies loss of integration of pathways controlling energy balance, the cell cycle, and apoptosis due to aberrant AMPK and p27 function as a feature of cells that have lost the Tsc2 tumor suppressor gene. [Cancer Res 2008;68(16):6496-506]

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The Tuberous Sclerosis Complex 2 (TSC2) gene product, tuberin, acts as a negative regulator of mTOR signaling, and loss of tuberin function leads to tumors of the brain, skin, kidney, heart and lungs. Previous studies have shown that loss of tuberin function affects the stability and subcellular localization of the cyclin-dependent kinase inhibitor (CKI) p27, although the mechanism(s) by which tuberin modulates p27 stability have not been elucidated. Previous studies have also shown that AMPK, which functions in an energy-sensing pathway in the cell, becomes activated in the absence of tuberin. Here we show that in Tsc2-null tumors and cell lines, AMPK activation correlates with an increase in p27 levels, and inhibition of AMPK signaling decreases p27 levels in these cells. In addition, activation of AMPK led to phosphorylation of p27 at the conserved terminal threonine residue of murine p27 (T197) in both in vitro kinase assays and in cells. Phosphorylation of p27 at T197 led to increased interaction between p27 and 14-3-3 proteins and increased the protein stability of p27. Furthermore, activation of AMPK signaling promoted the interaction between p27 and 14-3-3 proteins and increased the stability of the p27 protein in a manner that was dependent on T197. These data identify a conserved mechanism for regulation of p27 stability via phosphorylation at the terminal threonine (mT197/hT198), which when AMPK is activated, results in stabilization of the p27 protein.

Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.

Uterine leiomyom as, also called broids, are the most common reproductive tract neoplasm and the leading indication for hysterectomy in premenopausal wom en. The discovery and developme nt of medicinal therapies for uterine leiomyom a have been ham pered by a lack of understand ing regarding the etiology and molecular mechanisms underlying the developme nt of these lesions. Although the estrogen responsiveness of uterine leiomyom a is well established, the impact of environmental estrogen s and their contribution to the developm ent of these tumors is currently unknown . The Eker rat model of uterine leiomyom a has proven useful for addressing these issues and understand ing the pathophysiology of this disease. The Eker rat is the only animal m odel that develops spontaneou s uterine leiom yomas, and these tumors share m any characteristics with those found in hum ans. The availability of tumor-derived cell lines from these rats has m ade this a valuable in vitro/in vivo model system for experimen tal studies to investigate molecular m echanisms of disease and to design interventional and preventative strategies for this clinically relevan t tumor.Keywords. Estrogen; steroid; hormone; Tsc-2; rat; m yometrium ; xenoestrog en; uterus UTERINE LEIOMYOMAS ARE HORMONE RESPONSIVE Uterine leiomyomas, the benign smooth muscle tum ors originating from the myometrium and often calledbroids, are the m ost common reproductive tract neoplasm in women. These tumors have a reported incidence as high as 77% and are the leading indication for hysterectom y in the United States (7, 11). Com mon symptom s associated with these tumors are dysmenorrhea, m enorrhagia, infertility, and m orbidity (7). Although the incidence of this neoplasm is extrem ely high, the etiology has rem ained elusive. Many risk factors associated with broid development have been identi ed from epidem iologic studies, including obesity, age, race, and unopposed estrogen exposure (Figure 1). Some protective factors identi ed from these studies include the use of oral contraceptives, pregnancy, and cigarette smoking. In the majority of cases, alterations in hormonal m ilieu appear to underlie the impact of these risk factors (8-10, 22-25, 29 -31).The growth of uterine leiomyomas is thought to be modulated by the ovarian horm ones estrogen (E 2 ) and progesterone. The horm onal dependence of leiom yoma growth is indicated by the fact that m ost of these tumors are diagnosed during the reproductive years, increase in size during pregnancy, and regress after the onset of menopause (7), events that all coincide with changes in hormonal milieu. Other evidence supporting estrogen dependence of uterine leiomyomas are the increased estrogen receptor (ER) expression and decreased E 2 metabolism observed in these tumors (2). Consistent with hormones in uencing the growth of these tumors is the fact that gonadotropin-releasing hormone (GnRH) agonists, which interfere with signaling pathways of the hypothalamic- pituitary axis, halt or reverse uterine leiomyoma gr...

Tamoxifen, a selective estrogen receptor modulator, is a commonly prescribed adjuvant therapy for estrogen receptor-α (ERα)-positive breast cancer patients. To determine if extracellular factors contribute to the modulation of IGF-1 signaling after tamoxifen treatment, MCF-7 cells were treated with IGF-1 in conditioned medium (CM) obtained from 4-OHT-treated MCF-7 cells and the accumulation of phospho-Akt (S473) was measured. CM inhibited IGF-1-dependent cell signaling and suggesting the involvement of extracellular factors (ie. IGFBPs). A significant increase in IGFBP-1 mRNA and extracellular IGFBP-1 protein was observed in 4-OHT-treated MCF-7 cells. Knockdown experiments demonstrated that both GPER1 and CREB mediate IGFBP-1 induction. Furthermore, experiments showed that 4-OHT-dependent IGFBP-1 transcription is downstream of GPER1-activation in breast cancer cells. Additionally, neutralization and knockdown experiments demonstrated a role for IGFBP-1 in the observed inhibition of IGF-1 signaling. These results suggested that 4-OHT inhibits IGF-1 signaling via GPER1 and CREB mediated extracellular IGFBP-1 accumulation in breast cancer cells.

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