High calcium chemically defined culture medium

A high calcium chemically defined animal cell culture medium including vitamins A and D and a fatty acid or its ester. The medium is particularly adapted for the primary or secondary culture of epithelial cells. However, the medium may be utilized for establishing and maintaining cell lines, in particular myelomae and hybridomae.Board of Regents, University of Texas Syste

Tumor marker protein for cancer risk assessment

This invention relates to the isolation, identification and sequencing of a cancer associated protein, preparation of hybridization probes therefrom, preparation of antibodies thereto, and methods of cancer risk assessment and diagnosis.Board of Regents, University of Texas Syste

Activation of T-cell Protein-tyrosine Phosphatase Suppresses Keratinocyte Survival and Proliferation following UVB Irradiation

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.STAT3 promotes UVB-induced keratinocyte cell proliferation. Results TC-PTP inhibits STAT3-mediated mouse keratinocyte skin cell proliferation and survival following UVB irradiation. Conclusion TC-PTP plays an important role in the skin cell response to UVB radiation by regulating cell proliferation and survival. Significance TC-PTP shows tumor-suppressive capabilities in skin

UVB-induced nuclear translocation of TC-PTP by AKT/14-3-3σ axis inhibits keratinocyte survival and proliferation

Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation

The role of T-cell protein tyrosine phosphatase in epithelial carcinogenesis

T-cell protein tyrosine phosphatase (TC-PTP, encoded by PTPN2) is a non-receptor PTP that is mostly highly expressed in hematopoietic tissues. TC-PTP modulates a variety of physiological functions including cell cycle progression, cell survival and proliferation, and hematopoiesis through tyrosine dephosphorylation of its target substrates, such as EGFR, JAK1, JAK3, STAT1, and STAT3. Studies with whole or tissue-specific loss of TC-PTP function transgenic mice have shown that TC-PTP has crucial roles in the regulation of the immune response, insulin signaling, and oncogenic signaling. More recently, generation of epidermal-specific TC-PTP-deficient mice for use in multistage skin carcinogenesis bioassays demonstrated that TC-PTP suppresses skin tumor formation by negatively regulating STAT3 and AKT signaling. Further investigation showed that TC-PTP also minimizes UVB-induced epidermal cell damage by promoting apoptosis through the negative regulation of Flk-1/JNK signaling. These findings provide major evidence for a tumor suppressive function for TC-PTP against environment-induced skin cancer. Here, we will discuss TC-PTP, its substrates, and its functions with an emphasis on its role in skin carcinogenesis

4-Hydroxy-3-Methoxybenzoic Acid Methyl Ester: A Curcumin Derivative Targets Akt/NFκB Cell Survival Signaling Pathway: Potential for Prostate Cancer Management

AbstractTranscription factor NFKB and the serine/threonine kinase Akt play critical roles in mammalian cell survival signaling and have been shown to be activated in various malignancies including prostate cancer (PCA). We have developed an analogue of curcumin called 4hydroxy-3-methoxybenzoic acid methyl ester (HMBME) that targets the Akt/NFκB signaling pathway. Here, we demonstrate the ability of this novel compound to inhibit the proliferation of human and mouse PCA cells. HMBME-induced apoptosis in these cells was tested by using multiple biochemical approaches, in addition to morphological analysis. Overexpression of constitutively active Akt reversed the HMBME-induced growth inhibition and apoptosis, illustrating the direct role of Akt signaling in HMBME-mediated growth inhibition and apoptosis. Further, investigation of the molecular events associated with its action in LNCaP cells shows that: 1) HMBME reduces the level of activated form of Akt (phosphorylated Akt); and 2) inhibits the Akt kinase activity. Further, the transcriptional activity of NFκB, the DNA-binding activity of NFκB, and levels of p65 were all significantly reduced following treatment with HMBME. Overexpression of constitutively active Akt, but not the kinase dead mutant of Akt, activated the basal NFκB transcriptional activity. HMBME treatment had no influence on this constitutively active Aktaugmented NFκB transcriptional activity. These data indicate that HMBME-mediated inhibition of Akt kinase activity may have a potential in suppressing/decreasing the activity of major survival/antiapoptotic pathways. The potential use of HMBME as an agent that targets survival mechanisms in PCA cells is discussed

Targeted disruption of TC-PTP in the proliferative compartment augments STAT3 and AKT signaling and skin tumor development

Tyrosine phosphorylation is a vital mechanism that contributes to skin carcinogenesis. It is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Here, we report the critical role of T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, in chemically-induced skin carcinogenesis via the negative regulation of STAT3 and AKT signaling. Using epidermal specific TC-PTP knockout (K14Cre.Ptpn2fl/fl) mice, we demonstrate loss of TC-PTP led to a desensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP deficiency also resulted in a significant increase in epidermal thickness and hyperproliferation following exposure to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis showed that both phosphorylated STAT3 and phosphorylated AKT expressions were significantly increased in epidermis of TC-PTP-deficient mice compared to control mice following TPA treatment. Inhibition of STAT3 or AKT reversed the effects of TC-PTP deficiency on apoptosis and proliferation. Finally, TC-PTP knockout mice showed a shortened latency of tumorigenesis and significantly increased numbers of tumors during two-stage skin carcinogenesis. Our findings reveal that TC-PTP has potential as a novel target for the prevention of skin cancer through its role in the regulation of STAT3 and AKT signaling

Role of AMPK and PPARα in the anti-skin cancer effects of ursolic acid

The phytonutrient ursolic acid (UA), present in apples, rosemary, and other plant sources, has anti-cancer properties in a number of systems, including skin cancers. However, few reports have examined upstream mechanisms by which UA may prevent or treat cancer. Recent reports have indicated UA induces death of cancer cell lines via AMP-activated protein kinase (AMPK), an energy-sensing kinase which possesses both pro-metabolic and anti-cancer effects. Other studies have shown UA activates peroxisome proliferator activated receptor α (PPARα) and the glucocorticoid receptor (GR). Here, we found the cytotoxic effect of UA in skin carcinoma cells required AMPK activation. In addition, two inhibitors of PPARα partially reversed the cytotoxic effects of UA, suggesting its effects are at least partially mediated through this receptor. Finally, inhibition of the GR did not reverse the effects of UA nor did this compound bind the GR under the conditions of experiments performed. Overall, studies elucidating the anti-cancer effects of UA may allow for the development of more potent analogues utilizing similar mechanisms. These studies may also reveal the mediators of any possible side effects or resistance mechanisms to UA therapy

Inhibition of neddylation represses lipopolysaccharide-induced proinflammatory cytokine production in macrophage cells

Background: Lipopolysaccharides (LPSs) up-regulate proinflammatory cytokines in macrophages, partly through a NF-κB-dependent process. Results: Blocking neddylation, which helps regulate NF-κB, represses LPS-induced up-regulation of proinflammatory cytokines. Conclusion: Neddylation plays a role in the up-regulation of NF-κB-regulated proinflammatory cytokines produced by macrophages in response to LPS. Significance: Inhibition of neddylation represents a novel and effective method for the prevention of LPS-induced proinflammatory cytokines