Morphoproteomic analysis reveals an overexpressed and constitutively activated phospholipase D1-mTORC2 pathway in endometrial carcinoma.

The mammalian target of rapamycin (MTOR) assembles into two distinct complexes: mTOR complex 1 (mTORC1) is predominantly cytoplasmic and highly responsive to rapamycin, whereas mTOR complex 2 (mTORC2) is both cytoplasmic and nuclear, and relatively resistant to rapamycin. mTORC1 and mTORC2 phosphorylatively regulate their respective downstream effectors p70S6K/4EBP1, and Akt. The resulting activated mTOR pathways stimulate protein synthesis, cellular proliferation, and cell survival. Moreover, phospholipase D (PLD) and its product, phosphatidic acid (PA) have been implicated as one of the upstream activators of mTOR signaling. In this study, we investigated the activation status as well as the subcellular distribution of mTOR, and its upstream regulators and downstream effectors in endometrial carcinomas (ECa) and non-neoplastic endometrial control tissue. Our data show that the mTORC2 activity is selectively elevated in endometrial cancers as evidenced by a predominant nuclear localization of the activated form of mTOR (p-mTOR at Ser2448) in malignant epithelium, accompanied by overexpression of nuclear p-Akt (Ser473), as well as overexpression of vascular endothelial growth factor (VEGF)-A isoform, the latter a resultant of target gene activation by mTORC2 signaling via hypoxia-inducible factor (HIF)-2alpha. In addition, expression of PLD1, one of the two major isoforms of PLD in human, is increased in tumor epithelium. In summary, we demonstrate that the PLD1/PA-mTORC2 signal pathway is overactivated in endometrial carcinomas. This suggests that the rapamycin-insensitive mTORC2 pathway plays a major role in endometrial tumorigenesis and that therapies designed to target the phospholipase D pathway and components of the mTORC2 pathway should be efficacious against ECa.

The cyclotron production and nuclear imaging of bromine-77

In this investigation, bromine-77 was produced with a medical cyclotron and imaged with gamma cameras. Br-77 emits a 240 kev photon with a half life of 56 hours. The C-Br bond is stronger than the C-I bond and bromine is not collected in the thyroid. Bromine can be used to label many organic molecules by methods analogous to radioiodination. The only North American source of Br-77 in the 70's and 80's was Los Alamos National Laboratory, but it discontinued production in 1989. In this method, a p,3n reaction on Br-77 produces Kr-77 which decays with a 1.2 hour half life to Br-77. A cyclotron generated 40 MeV proton beam is incident on a nearly saturated NaBr or LiBr solution contained in a copper or titanium target. A cooling chamber through which helium gas is flowed separates the solution from the cyclotron beam line. Helium gas is also flowed through the solution to extract Kr-77 gas. The mixture flows through a nitrogen trap where Kr-77 freezes and is allowed to decay to Br-77. Eight production runs were performed, three with a copper target and five with a titanium target with yields of 40, 104, 180, 679, 1080, 685, 762 and 118 uCi respectively. Gamma ray spectroscopy has shown the product to be very pure, however corrosion has been a major obstacle, causing the premature retirement of the copper target. Phantom and in-vivo rat nuclear images, and an autoradiograph in a rat are presented. The quality of the nuclear scans is reasonable and the autoradiograph reveals high isotope uptake in the renal parenchyma, a more moderate but uniform uptake in pulmonary and hepatic tissue, and low soft tissue uptake. There is no isotope uptake in the brain or the gastric mucosa. ^

Transcriptional upregulation of the p21Cip1 promoter by STAT3 and nuclear ErbB2

Over-expression of the receptor tyrosine kinase ErbB2 is prevalent in approximately 30% of human breast carcinomas and confers Taxol resistance. In breast cancer cells, Taxol induces tubulin polymerization and hyperstable microtubule formation. This in turn prematurely activates Cdc2 kinase allowing early entry into the G2/M phase of the cell cycle resultant in mitotic catastrophe followed by apoptosis. Over-expression of ErbB2 upregulates p21Cip1, which inhibits Cdc2 activation, and leads to Taxol resistance in patients. However, the mechanism of ErbB2-mediated p21 Cip1 upregulation is unclear. Here in this study, we investigated the mechanism of ErbB2 downstream signaling events leading to upregulation. The CDKN1A (p21Cip1) gene promoter contains numerous cis-elements including a Signal transducer and activator of transcription (STAT) Inducable Element (SIE) located at -679 kb. Our studies showed ErbB2 overexpressing cells had increased activated levels of STAT3, and therefore we hypothesized that STAT3 is responsible for the upregulation of the p21Cip1 promoter by ErbB2. EMSA and ChIP assays confirmed the binding of STAT3 to the p21Cip1 promoter and luciferase assays showed higher p21 Cip1 promoter activity in ErbB2 over-expressing transfectants when compared to parental cells, in a STAT3 binding site dependant manner. Additionally, reduced level of STAT3 led to reduced p21Cip1 protein expression and promoter activity indicating that both the STAT3 binding site and STAT3 protein are required for ErbB2-mediated p21Cip1 upregulation. Further investigation of ErbB2 downstream signaling showed increased Src kinase activity in ErbB2 over-expressing cells which was required for ErbB2-mediated STAT3 activation and p21Cip1 increase. Treatment of ErbB2 over-expressing resistant cells with STAT3 inhibitor peptides sensitized the cells to Taxol. In addition to classical signal transduction pathways, I identified a novel ErbB2 mediated regulatory mechanism of p21Cip1. I found that a nuclear ErbB2 and STAT3 complex binds directly to the p21Cip1 promoter offering a non-classical mechanism of p21Cip1 promoter regulation. These data suggest that ErbB2 over-expression can confer Taxol resistance of breast cancer cells by transcriptional upregulation of p21 Cip1 via activation of STAT3 by Src kinase and also by cooperation with nuclear ErbB2. The data suggest a potential clinical mechanism for STAT3 inhibitors in sensitizing ErbB2 over-expressing breast cancers to Taxol. ^

Syntaxin 6- and microtubule- mediated intracellular trafficking contributes to Golgi and nuclear translocation of EGFR

Receptor-mediated endocytosis is well known for its degradation and recycling trafficking. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER), and the nucleus to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the mechanism of intracellular trafficking from cell surface to the Golgi is not yet completed understood. Here we report a mechanism of Golgi translocation of EGFR in which EGF-induced EGFR travels to the Golgi via microtubule (MT)-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6 (Syn6)-mediated membrane fusion. We also demonstrate that the Golgi translocation of EGFR is necessary for its consequent nuclear translocation and transcriptional activity. Interestingly, foreign protein such as bacterial cholera toxin, which is known to activate its pathological function through the Golgi/ER retrograde pathway, also utilizes the MT/Syn6 pathway. Thus, the MT, and syntaxin 6 mediated trafficking pathway from cell surface to the Golgi and ER defines a comprehensive retrograde trafficking route for both cellular and foreign molecules to travel from cell surface to the Golgi and the nucleus.

The role of the conserved N -terminal domain of STAT3 in STAT3 dephosphorylation and nuclear export

Rapid redistribution of STAT subcellular localization is an essential feature of cytokine signaling. To elucidate the molecular basis of STAT3 function, which plays a critical role in controlling innate immune responses in vivo, we initiated studies to determine the mechanisms controlling STAT3 nuclear trafficking. We found that STAT3 is transported to the nucleus in the absence of cytokine treatment, as judged by indirect immunofluorescence studies in the presence of leptomycin B, an inhibitor of CRM1-dependent nuclear export, suggesting that the non-phosphorylated STAT3 protein contains a functional nuclear import signal. An isoform lacking the STAT3 N-terminal domain (Δ133STAT3) retains the ability to undergo constitutive nuclear localization, indicating that this region is not essential for cytokine-independent nuclear import. Δ133STAT3 is also transported to the nucleus following stimulation with interleukin-6 (IL-6). Interestingly, IL-6-dependent tyrosine phosphorylation of Δ133STAT3 appears to be prolonged and the nuclear export of the protein delayed in cells expressing endogenous STAT3, consistent with defective Δ133STAT3 dephosphorylation. Endogenous STAT3 does not promote the nuclear export of Δ133STAT3, although dimerization between endogenous Stat3 and Δ133STAT3 is detected readily. Thus, the STAT3 N-terminal domain is not required for dimerization with full-length STAT3, yet appears to play a role in proper export of Stat3 from the nucleus following cytokine stimulation. STAT3-deficient cells reconstituted with Δ133STAT3 show enhanced and prolonged Stat1 signaling in response to IL-6, suggesting that induction of the STAT3-dependent negative regulator SOCS3 is impaired. In fact, Δ133STAT3 fails to induce SOCS3 mRNA efficiently. These studies collectively indicate that the STAT3 N-terminal region may be important for IL-6-dependent target gene activation and nuclear dephosphorylation, while dispensable for nuclear import. STAT3 is an oncogene. STAT3 is constitutively activated in primary tumors of many types. Thus far, research in the design of STAT3 protein inhibitors has focused on the SH2 and DNA-binding domains of STAT3. Interference with these domains eliminates all signaling through STAT3. If the N-terminal domain is involved in tetramerization on a subset of target genes, inhibition of this region may lead to a more selective inhibition of some STAT3 functions while leaving others intact. ^

THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

NHERF1 – New Modifier of Colorectal Cancer Progression

Colorectal cancer (CRC) develops from multiple progressive modifications of normal intestinal epithelium into adenocarcinoma. Loss of cell polarity has been implicated as an early event in this process, but the molecular players involved are not well known. NHERF1 (Na+/H+ Exchanger Regulatory Factor 1) is an adaptor protein with apical membrane localization in polarized epithelia. In this study, we tested our hypothesis that NHERF1 plays a role in CRC. We examined surgical CRC resection specimens for changes in NHERF1 expression, and modeled these changes in two- and three-dimensional (2D and 3D) Caco-2 CRC cell systems. NHERF1 had significant alterations from normal to adenoma and carcinoma transitions (2=38.5, d.f.=4, P<0.001), displaying apical membrane localization in normal tissue but loss of expression in adenoma and ectopic overexpression in carcinoma. In Caco-2 cell models, NHERF1 depletion induced epithelial-mesenchymal-transition in 2D cell monolayers and disruption of apical-basal polarity in 3D cyst system. The mesenchymal phenotype of NHERF1-depleted cells was fully restored by re-expression of NHERF1 at the apical membrane. Cytoplasmic and nuclear NHERF1 re-expression not only failed to restore the epithelial phenotype but led to more aggressive phenotypes. Our findings suggest that membrane NHERF1 is an important regulator of epithelial morphogenesis, and that changes in NHERF1 expression correlate with CRC progression. NHERF1 loss and ectopic expression that induce massive disruption of epithelial cell polarity may, thereby, mark important steps in CRC development.

AN ANALYSIS OF THE DNA DAMAGE INDUCED BY NICKEL COMPOUNDS IN CHINESE HAMSTER OVARY CELLS (CARCINOGENESIS, HETEROCHROMATIN, CYTOTOXICITY, REPLICATION, PROTEIN CROSSLINKING)

The carcinogenic activity of water-insoluble crystalline nickel sulfide requires phagocytosis and lysosome-mediated intracellular dissolution of the particles to yield Ni('2+). This study investigated the extent and nature of the DNA damage in Chinese hamster ovary cells treated with various nickel compounds using the technique of alkaline elution. Crystalline NiS and water-soluble NiCl(,2) induced single strand breaks that were repaired quickly and DNA-protein crosslinks that persisted up to 24 hr after exposure to nickel. The induction of single strand breaks was concentration dependent at both noncytotoxic and lethal amounts of nickel. The induction of DNA-protein crosslinks was concentration dependent but was absent at lethal amounts of nickel. The cytoplasmic and nuclear uptake of nickel was concentration dependent even at the toxic level of nickel. However, the induction of DNA-protein crosslinks by nickel required active cell cycling and occurred predominantly in mid-late S phase of the cell cycle, suggesting that the lethal amounts of nickel inhibited DNA-protein crosslinking by inhibiting active cell cycling. Since the DNA-protein crosslinking induced by nickel was resistant to DNA repair, the nature of this lesion was investigated using various methods of DNA isolation and chromatin fractionation in combination with SDS-polyacrylamide gel electrophoresis. High molecular weight, non-histone chromosomal proteins and possibly histone 1 were preferentially crosslinked to DNA by nickel. The crosslinked proteins were concentrated in a magnesium-insoluble fraction of sonicated chromatin (5% of the total) that was similar to heterochromatin in solubility and protein composition. Alterations in DNA structure and function, brought about by the effect of nickel on protein-DNA interactions, may be related to the carcinogenicity of nickel compounds. ^

Morphoproteomic evidence of constitutively activated and overexpressed mTOR pathway in cervical squamous carcinoma and high grade squamous intraepithelial lesions.

Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.

DEVELOPMENTAL AND CELLULAR FUNCTIONS OF DELTA-CATENIN

Catenins have diverse and powerful roles in embryogenesis, homeostasis or disease progression, as best exemplified by the well-known beta-catenin. The less studied delta-catenin likewise contains a central Armadillo-domain. In common with other p120 sub-class members, it acts in a variety of intracellular compartments and modulates cadherin stability, small GTPase activities and gene transcription. In mammals, delta-catenin exhibits neural specific expression, with its knock-out in mice correspondingly producing cognitive defects and synaptic dysfunctions. My work instead employed the amphibian, Xenopus laevis, to explore delta-catenin’s physiological functions in a distinct vertebrate system. Initial isolation and characterization indicated delta-catenin’s expression in Xenopus. Unlike the pattern observed for mammals, delta-catenin was detected in most adult Xenopus tissues, although enriched in embryonic structures of neural fate as visualized using RNA in-situ hybridization. To determine delta-catenin’s requirement in amphibian development, I employed anti-sense morpholinos to knock-down gene products, finding that delta-catenin depletion results in developmental defects in gastrulation, neural crest migration and kidney tubulogenesis, phenotypes that were specific based upon rescue experiments. In biochemical and cellular assays, delta-catenin knock-down reduced cadherin levels and cell adhesion, and impaired activation of RhoA and Rac1, small GTPases that regulate actin dynamics and morphogenetic movements. Indeed, exogenous C-cadherin, or dominant-negative RhoA or dominant-active Rac1, significantly rescued delta-catenin depletion. Thus, my results indicate delta-catenin’s essential roles in Xenopus development, with contributing functional links to cadherins and Rho family small G proteins. In examining delta-catenin’s nuclear roles, I identified delta-catenin as an interacting partner and substrate of the caspase-3 protease, which plays critical roles in apoptotic as well as non-apoptotic processes. Delta-catenin’s interaction with and sensitivity to caspase-3 was confirmed using assays involving its cleavage in vitro, as well as within Xenopus apoptotic extracts or mammalian cell lines. The cleavage site, a highly conserved caspase consensus motif (DELD) within Armadillo-repeat 6 of delta-catenin, was identified through peptide sequencing. Cleavage thus generates an amino- (1-816) and carboxyl-terminal (817-1314) fragment each containing about half of the central Armadillo-domain. I found that cleavage of delta-catenin both abolishes its association with cadherins, and impairs its ability to modulate small GTPases. Interestingly, the carboxyl-terminal fragment (817-1314) possesses a conserved putative nuclear localization signal that I found is needed to facilitate delta-catenin’s nuclear targeting. To probe for novel nuclear roles of delta-catenin, I performed yeast two-hybrid screening of a mouse brain cDNA library, resolving and then validating its interaction with an uncharacterized KRAB family zinc finger protein I named ZIFCAT. My results indicate that ZIFCAT is nuclear, and suggest that it may associate with DNA as a transcriptional repressor. I further determined that other p120 sub-class catenins are similarly cleaved by caspase-3, and likewise bind ZIFCAT. These findings potentially reveal a simple yet novel signaling pathway based upon caspase-3 cleavage of p120 sub-family members, facilitating the coordinate modulation of cadherins, small GTPases and nuclear functions. Together, my work suggested delta-catenin’s essential roles in Xenopus development, and has revealed its novel contributions to cell junctions (via cadherins), cytoskeleton (via small G proteins), and nucleus (via ZIFCAT). Future questions include the larger role and gene targets of delta-catenin in nucleus, and identification of upstream signaling events controlling delta-catenin’s activities in development or disease progression.

An examination of the characteristics, concentration and distribution of androgen receptor in rat testis during sexual maturation

Previously reported androgen receptor concentrations in rat testis and testicular cell types have varied widely. In the studies reported here a nuclear exchange assay was established in rat testis in which exchange after 86 hours at 4$\sp\circ$C was greater than 85% complete and receptor was stable. Receptor concentration per DNA measured by exchange declined between 15 and 25 days of age in the rat testis, then increased 4-fold during sexual maturation. Proliferation of germ cells which had low receptor concentration appeared to account for the early decline in testicular receptor concentration, whereas increase in receptor number per Sertoli cell between 25 and 35 days of age contributed to the later increase. Increase in Leydig cell number during maturation appeared to account for the remainder of the increase due to the high receptor concentration in these cells. Detailed studies showed that other possible explanations for changes in receptor number (e.g. shifts in receptor concentration between the cytosol and nuclear subcellular compartments or changes in the affinity of the receptor for its ligands) were not likely.^ Androgen receptor dynamics in testicular cells showed rapid, specific uptake of ($\sp3$H) -testosterone that was easily blocked by unlabeled testosterone (RA of 7 nM in both cell types), and medroxyprogesterone acetate (RA of 28 and 16 nM in Sertoli and peritubular cells, respectively), but not as well by the anti-androgens cyproterone acetate (RA of 116 and 68 nM) and hydroxyflutamide (RA of 300 and 180 nM). The affinity of the receptor for the ligand dimethylnortestosterone was similar in the two cell types (K$\rm\sb{d}$ values of 0.78 and 0.71 nM for Sertoli and peritubular cells) and was virtually identical with the affinity of the whole testis receptor (0.89 nM). Medroxyprogesterone acetate and testosterone significantly increased nuclear androgen receptor concentration relative to untreated controls in Sertoli and peritubular cells, whereas hydroxyflutamide and cyproterone acetate did not. Despite the different embryological origins of peritubular and Sertoli cells, their responses to both androgens and anti-androgens were similar. In addition, these studies suggest that peritubular cells are as likely as Sertoli cells to be primary androgen targets. ^

Expression and function of $\beta$-tubulin isotypes in Chinese hamster ovary (CHO) cells

The availability of isotype specific antisera for $\beta$-tubulin, coupled with genetic and biochemical analysis, has allowed the determination of $\beta$-tubulin isotype expression and distribution in Chinese hamster ovary (CHO) cells. Using genetic manipulations involving selection for colcemid resistance followed by reversion and reselection for drug resistance, we have succeeded in isolating cell lines that exhibit three major and one minor $\beta$-tubulin spots by two-dimensional gel electrophoresis. In concert with isotype specific antibodies, analysis of these mutants demonstrates that CHO cells express two copies of isotype I, at least one copy of isotype IV, and very small amounts of isotype V. Their stoichiometry is approximately 1:1:0.7:0.2. All three isotypes assemble into both cytoplasmic and spindle microtubules, and are similar in their responses to cold, colcemid, and calcium induced depolymerization. They have comparable turnover rates and are equally sensitive to depression of synthesis upon colchicine treatment. These results suggest that $\beta$-tubulin isotypes are used interchangeably to assemble microtubule structures in CHO cells. However, of 18 colcemid resistant mutants with a demonstrable alteration in $\beta$-tubulin, all were found to have the alteration in isotype I, thus leaving open the possibility that subtle differences in isotype properties may exist. Under various conditions of the cell growth, the relative proportion of each expressed isotype does not significantly seem to change except in the early G1 phase of the cell cycle. At this time the synthesis of isotype V increases more than two fold relative to isotype I and IV, while at the same time, total $\beta$-tubulin synthesis is decreased about 60-70%. ^

Molecular analysis of $\beta$1,4-galactosyl-transferase localization to the cell surface and participation in cell adhesion

$\beta$1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is found in two subcellular compartments where it performs different functions. In the trans-Golgi complex, GalTase participates in oligosaccharide biosynthesis as do other glycosyltransferases. GalTase is also found on the cell surface, where it associates with the cytoskeleton and functions as a receptor for extracellular oligosaccharide ligands. Although we know much regarding GalTase function on the cell surface, little is known about the mechanisms underlying its transport to the plasma membrane. Cloning of the GalTase gene revealed that there are two GalTase proteins (i.e., long and short) with different size cytoplasmic tails. This raises the possibility that differences in the cytoplasmic domain of GalTase may influence its subcellular distribution. The object of this study was to examine this hypothesis directly through the use of molecular, immunological, and biochemical approaches.^ To examine whether the two GalTase proteins are targeted to different subcellular compartments, F9 embryonal carcinoma cells were transfected with either long or short GalTase cDNAs and intracellular and cell surface enzyme levels measured. Cell surface GalTase activity was enriched in cells overexpressing the long, but not the form of short GalTase. Furthermore, a dominant negative mutation in cell surface GalTase was created by transfecting cells with GalTase cDNAs encoding a truncated version of long GalTase devoid of the extracellular catalytic domain. Overexpressing the complete cytoplasmic and transmembrane domains of long GalTase led to a loss of GalTase-dependent cellular adhesion by specifically displacing surface GalTase from its cytoskeletal associations. In contrast, overexpressing the analogous truncated protein of short GalTase had no effect on cell adhesion. Finally, chloramphenicol acetyltransferase (CAT) reporter proteins were used to determine directly whether the cytoplasmic domains of long and short GalTase were responsible for differential subcellular distribution. The cytoplasmic and transmembrane domains of long GalTase led to CAT expression on the ceil surface and its association with the detergent-insoluble cytoskeleton; the analogous fusion protein containing short GalTase was restricted to the Golgi compartment. These results suggest that the cytoplasmic domain unique to long GalTase is responsible for targeting a portion of this protein to the cell surface and associating it with the cytoskeleton, enabling it to function as a cell adhesion molecule. ^

Analysis of the mechanisms that maintain coordinate production of $\alpha$- and $\beta$-tubulin in mammalian cells

Alpha and beta tubulin are essential proteins in all eukaryotic cells. To study how cells maintain coordinate levels of these two interacting proteins, we have used PCR to add a 9 amino acid epitope from influenza hemagglutinin protein onto the carboxyl terminus of $\alpha$1 and $\beta$1-tubulin. The chimeric tubulin genes (HA$\alpha$1 and HA$\beta$1) were transfected into CHO cells and cell lines that stably express each gene were selected. Cells transfected with HA-tubulin do not exhibit any gross changes in growth or morphology. Immunofluorescence analysis demonstrated that HA-tubulins incorporate into both cytoplasmic and spindle microtubules. A quantitative biochemical assay was used to show that HA-tubulins incorporate into microtubules to a normal extent and do not alter the steady state distribution of endogenous tubulin between monomer and polymer pools. Two-dimensional gel analysis of pulse-labeled cells indicated that when HA$\beta$1-tubulin is expressed at high levels, it slightly represses the synthesis of the endogenous $\beta$-tubulin but produces a small increase in the synthesis of $\alpha$-tubulin. Analysis of cells labeled to steady state showed that HA$\beta$1-tubulin accumulates to a similar level as the wild-type gene product, but together these polypeptides produce only a small increase in total tubulin content consistent with the increased synthesis of $\alpha$-tubulin. It thus appears that HA$\beta$1-tubulin successfully competes with endogenous $\beta$-tubulin for heterodimer formation and that free $\beta$-tubulin subunits (endogenous and HA$\beta$1) are selectively degraded to maintain coordinate amounts of $\alpha$- and $\beta$-tubulin. In addition, the increased synthesis of $\alpha$-tubulin suggested the existence of a mechanism to ensure coordinate synthesis of $\alpha$- and $\beta$-tubulin subunits. To analyze whether reciprocal changes in endogenous tubulin synthesis occur when $\alpha$-tubulin is overexpressed, stably transfected CHO cell lines were isolated in which HA$\alpha$1-tubulin represents 50% of the total $\alpha$-tubulin, and its relative abundance can be further increased to 85-90% by treatment with sodium butyrate. In contrast with results obtained using HA$\beta$1-tubulin, transfection of HA$\alpha$1-tubulin decreased the synthesis of endogenous $\alpha$-tubulin to 60% of normal with little or no change in $\beta$-tubulin synthesis. When the transfected cells were treated with sodium butyrate to further increase HA$\beta$1-tubulin production, a larger decrease in the synthesis of endogenous $\alpha$-tubulin (to 30% of normal) was observed. The repression on the synthesis of endogenous $\alpha$-tubulin polypeptide was found to be directly proportional to the expression of HA$\alpha$1-tubulin indicating the existence of an autoregulatory loop, where $\alpha$-tubulin inhibits its own synthesis. To determine whether overproduction of HA$\alpha$1-tubulin affected the transcription, message stability or translation of endogenous $\alpha$-tubulin, the steady state levels of $\alpha$-tubulin mRNA were analyzed by ribonuclease protection assays. The results showed that the steady state level of $\alpha$-tubulin mRNA is not affected by the overexpression of HA$\alpha$1-tubulin, indicating that the repression is translational. The results are compatible with a model in which $\beta$-tubulin synthesis is largely unperturbed by overexpression of other tubulin subunits, and excess $\beta$-tubulin subunits are rapidly degraded to maintain coordinate $\alpha$- and $\beta$-tubulin levels at steady state. In contrast, free $\alpha$-tubulin represses its own synthesis at the translational level, suggesting that its level of production may be controlled by the amount of $\beta$-tubulin available for heterodimer formation. ^

The role of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) in normal human prostate epithelial cells and prostate cancer development

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial (NHP) cells. Western blotting with multiple NHP cell strains and prostate cancer (PCa) cell lines reveals that the expression of 15-LOX2 is lost in all PCa cell lines, accompanied by decreased enzymatic activity. 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 are mostly excluded from the nucleus. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor arachidonic acid-metabolizing activity. Interestingly, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. When orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Finally, cultured NHP cells lose the expression of putative stem/progenitor cell markers, slow down in proliferation, and enter senescence. Several pieces of evidence implicate 15-LOX2 plays a role in replicative senescence of NHP cells: (1) promoter activity and the mRNA and protein levels of 15-LOX2 and its splice variants are upregulated in serially passaged NHP cells, which precede replicative senescence and occur in a cell-autonomous manner; (2) PCa cells stably expressing 15-LOX2 or 15-LOX2sv-b show a passage-related senescence-like phenotype; (3) enforced expression of 15-LOX2 or 15-LOX2sv-b in young NHP cells induce partial cell-cycle arrest and senescence-like phenotypes. Together, these results suggest that 15-LOX2 suppress prostate tumor development and do not necessarily depend on arachidonic acid-metabolizing activity and nuclear localization. Also, 15-LOX2 may serve as an endogenous prostate senescence gene and its tumor-suppressing functions might be associated with its ability to induce cell senescence. ^