Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts

Prostate stem-cell antigen (PSCA) is a cell-surface antigen expressed in normal prostate and overexpressed in prostate cancer tissues. PSCA expression is detected in over 80% of patients with local disease, and elevated levels of PSCA are correlated with increased tumor stage, grade, and androgen independence, including high expression in bone metastases. We evaluated the therapeutic efficacy of anti-PSCA mAbs in human prostate cancer xenograft mouse models by using the androgen-dependent LAPC-9 xenograft and the androgen-independent recombinant cell line PC3-PSCA. Two different anti-PSCA mAbs, 1G8 (IgG1κ) and 3C5 (IgG2aκ), inhibited formation of s.c. and orthotopic xenograft tumors in a dose-dependent manner. Furthermore, administration of anti-PSCA mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies suggest PSCA as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-PSCA mAbs for the treatment of local and metastatic prostate cancer.

Enhancement of translation by the epsilon element is independent of the sequence of the 460 region of 16S rRNA

The epsilon enhancer element is a pyrimidine-rich sequence that increases expression of T7 gene 10 and a number of Escherichia coli mRNAs during initiation of translation and inhibits expression of the recF mRNA during elongation. Based on its complementarity to the 460 region of 16S rRNA, it has been proposed that epsilon exerts its enhancer activity by base pairing to this complementary rRNA sequence. We have tested this model of enhancer action by constructing mutations in the 460 region of 16S rRNA and examining expression of epsilon-containing CAT reporter genes and recF–lacZ fusions in strains expressing the mutant rRNAs. Replacement of the 460 E.coli stem–loop with that of Salmonella enterica serovar Typhimurium or a stem–loop containing a reversal of all 8 bp in the helical region produced fully functional rRNAs with no apparent effect on cell growth or expression of any epsilon-containing mRNA. Our experiments confirm the reported effects of the epsilon elements on gene expression but show that these effects are independent of the sequence of the 460 region of 16S rRNA, indicating that epsilon–rRNA base pairing does not occur.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor β receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Extracellular matrix composition determines the transcriptional response to epidermal growth factor receptor activation

The transcriptional response to epidermal growth factor (EGF) was examined in a cultured cell model of adhesion. Gene expression was monitored in human embryonic kidney cells (HEK293) after attachment of cells to the extracellular matrix (ECM) proteins, laminin, and fibronectin, by using complementary DNA micorarrays printed with 1,718 individual human genes. Cluster analysis revealed that the influence of EGF on gene expression, either positive or negative, was largely independent of ECM composition. However, clusters of EGF-regulated genes were identified that were diagnostic of the type of ECM proteins to which cells were attached. In these clusters, attachment of cells to a laminin or fibronectin substrata specifically modified the direction of gene expression changes in response to EGF stimulation. For example, in HEK293 cells attached to fibronectin, EGF stimulated an increase in the expression of some genes; however, genes in the same group were nonresponsive or even suppressed in cells attached to laminin. Many of the genes regulated by EGF and ECM proteins in this manner are involved in ECM and cytoskeletal architecture, protein synthesis, and cell cycle control, indicating that cell responses to EGF stimulation can be dramatically affected by ECM composition.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-β. The ability to maintain growth in TGF-β was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-β resistance may explain our previously described gain of TGF-β resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-β resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

Separating Growth from Elastic Deformation during Cell Enlargement1

Plants change size by deforming reversibly (elastically) whenever turgor pressure changes, and by growing. The elastic deformation is independent of growth because it occurs in nongrowing cells. Its occurrence with growth has prevented growth from being observed alone. We investigated whether the two processes could be separated in internode cells of Chara corallina Klien ex Willd., em R.D.W. by injecting or removing cell solution with a pressure probe to change turgor while the cell length was continuously measured. Cell size changed immediately when turgor changed, and growth rates appeared to be altered. Low temperature eliminated growth but did not alter the elastic effects. This allowed elastic deformation measured at low temperature to be subtracted from elongation at warm temperature in the same cell. After the subtraction, growth alone could be observed for the first time. Alterations in turgor caused growth to change rapidly to a new, steady rate with no evidence of rapid adjustments in wall properties. This turgor response, together with the marked sensitivity of growth to temperature, suggested that the growth rate was not controlled by inert polymer extension but rather by biochemical reactions that include a turgor-sensitive step.

Adventitious Root Growth and Cell-Cycle Induction in Deepwater Rice1

Deepwater rice (Oryza sativa) is adapted to survive conditions of severe flooding over extended periods of time. During such periods adventitious roots develop to provide water, nutrients, and anchorage. In the present study the growth of adventitious roots was induced by treatment with ethylene but not auxin, cytokinin, or gibberellin. Root elongation was enhanced between 8 and 10 h after submergence. The population of cells in the S phase and expression of the S-phase-specific histone H3 gene increased within 4 to 6 h. Within 6 to 8 h the G2-phase population increased. Cell-cycle activation was accompanied by sequential induction of a cdc2-activating kinase homolog, R2, of two cdc2 genes, cdc2Os-1 and cdc2Os-2, and of three cyclin genes, cycA1;3, cycB2;1, and cycB2;2, but only induction of the R2 gene expression preceded the induction of the S phase, possibly contributing to cell-cycle regulation in the G1 phase. Both cdc2 genes were expressed at slightly higher levels during DNA replication. Transcripts of the A-type cyclin accumulated during the S and G2 phases, and transcripts of the B-type cyclins accumulated during the G2 phase. Cyclin expression was induced at all nodes with a similar time course, suggesting that ethylene acts systemically and that root primordia respond to ethylene at an early developmental stage.

Stat1-independent regulation of gene expression in response to IFN-γ

Although Stat1 is essential for cells to respond fully to IFN-γ, there is substantial evidence that, in the absence of Stat1, IFN-γ can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-γ in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-γ in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-γ, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-γ in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-γ receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-γ, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling.

Biologic consequences of Stat1-independent IFN signaling

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.

Genetic programs of epithelial cell plasticity directed by transforming growth factor-β

Epithelial–mesenchymal transitions (EMTs) are an essential manifestation of epithelial cell plasticity during morphogenesis, wound healing, and tumor progression. Transforming growth factor-β (TGF-β) modulates epithelial plasticity in these physiological contexts by inducing EMT. Here we report a transcriptome screen of genetic programs of TGF-β-induced EMT in human keratinocytes and propose functional roles for extracellular response kinase (ERK) mitogen-activated protein kinase signaling in cell motility and disruption of adherens junctions. We used DNA arrays of 16,580 human cDNAs to identify 728 known genes regulated by TGF-β within 4 hours after treatment. TGF-β-stimulated ERK signaling mediated regulation of 80 target genes not previously associated with this pathway. This subset is enriched for genes with defined roles in cell–matrix interactions, cell motility, and endocytosis. ERK-independent genetic programs underlying the onset of EMT involve key pathways and regulators of epithelial dedifferentiation, undifferentiated transitional and mesenchymal progenitor phenotypes, and mediators of cytoskeletal reorganization. The gene expression profiling approach delineates complex context-dependent signaling pathways and transcriptional events that determine epithelial cell plasticity controlled by TGF-β. Investigation of the identified pathways and genes will advance the understanding of molecular mechanisms that underlie tumor invasiveness and metastasis.

Identification of Two Type V Myosins in Fission Yeast, One of Which Functions in Polarized Cell Growth and Moves Rapidly in the Cell

We characterized the novel Schizosaccharomyces pombe genes myo4+ and myo5+, both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.

Evidence for a circulating islet cell growth factor in insulin-resistant states

Insulin resistance is a feature of many common disorders including obesity and type 2 diabetes mellitus. In these disorders, the β-cells compensate for the insulin resistance for long periods of time with an increase in secretory capacity, an increase in β-cell mass, or both. To determine whether the β-cell response might relate to a circulating growth factor, we have transplanted normal islets under the kidney capsule of normoglycemic insulin-resistant mice with two different models of insulin resistance: lean mice that have a double heterozygous deletion of the insulin receptor and insulin receptor substrate-1 (DH) or the obese, hyperglycemic ob/ob mice. In the grafts transplanted into both hosts, there was a marked increase in β-cell mitotic activity and islet mass that was comparable with that observed in the endogenous pancreas. By contrast, islets of the DH mouse transplanted into normal mice showed reduced mitotic index. These data suggest the insulin resistance is associated with a circulating islet cell growth factor that is independent of glucose and obesity.

Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism

The Ser/Thr kinase Raf-1 is a protooncogene product that is a central component in many signaling pathways involved in normal cell growth and oncogenic transformation. Upon activation, Raf-1 phosphorylates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs), leading to the propagation of signals. Depending on specific stimuli and cellular environment, the Raf-1–MEK–ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis. Here, we describe a MEK–ERK-independent prosurvival function of Raf-1. We found that Raf-1 interacts with the proapoptotic, stress-activated protein kinase ASK1 (apoptosis signal-regulating kinase 1) in vitro and in vivo. Deletion analysis localized the Raf-1 binding site to the N-terminal regulatory fragment of ASK1. This interaction allows Raf-1 to act independently of the MEK–ERK pathway to inhibit apoptosis. Furthermore, catalytically inactive forms of Raf-1 can mimic the wild-type effect, raising the possibility of a kinase-independent function of Raf-1. Thus, Raf-1 may promote cell survival through its protein–protein interactions in addition to its established MEK kinase function.

Physiological coupling of growth factor and steroid receptor signaling pathways: estrogen receptor knockout mice lack estrogen-like response to epidermal growth factor.

Past studies have shown that epidermal growth factor (EGF) is able to mimic the uterotropic effects of estrogen in the rodent. These studies have suggested a "cross-talk" model in which EGF receptor (EGF-R) signaling results in activation of nuclear estrogen receptor (ER) and its target genes in an estrogen-independent manner. Furthermore, in vitro studies have indicated the requirement for ER in this mechanism. To verify the requirement for ER in an in vivo system, EGF effects were studied in the uteri of ER knockout (ERKO) mice, which lack functional ER. The EGF-R levels, autophosphorylation, and c-fos induction were observed at equivalent levels in both genotypes indicating that removal of ER did not disrupt the EGF responses. Induction of DNA synthesis and the progesterone receptor gene in the uterus were measured after EGF treatment of both ERKO and wild-type animals. Wild-type mice showed increases of 4.3-fold in DNA synthesis, as well as an increase in PR mRNA after EGF treatment. However, these responses were absent in ERKO mice, confirming that the estrogen-like effects of EGF in the mouse uterus do indeed require the ER. These data conclusively demonstrate the coupling of EGF and ER signaling pathways in the rodent reproductive tract.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.