Adenosine 5'-triphosphate is known to function as a potent extracellular messenger, producing its effects via a distinct family of cell surface receptors. Different receptor subtypes have been shown to modulate different cellular functions such as proliferation, differentiation and apoptosis. We have investigated the functional expression and apoptotic action of the P2 X (7) receptor in human malignant melanoma tissue and cells. Incubation of cells with the potent P2 X (7) receptor agonist 2'-3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate leads to a decrease in cell number, which is dose-dependent and reversible by the antagonist 1-N,O-bis-[5-isoquinoline-sulfonyl]-N-methyl-L-tyrosyl)-4-phenyl-piperazine. Synthesis of the P2 X(7) receptor by these cells has been established by reverse transcriptase-polymerase chain reaction, immunohistochemistry, immunocytochemistry and cellular accumulation of the fluorescent DNA-binding dye YO-PRO-1. The P2 X(7) receptors have been shown to mediate apoptotic actions of extracellular nucleotides and represent a novel target for melanoma therapy.
Athymic mice, injected with A375 human melanoma cells, were treated daily with intraperitoneal injections of adenosine 5′-triphosphate (ATP). The tumour volume and animal weight were measured over the course of the experiment and the final tumour nodule weight was measured at the end of the experiment. Tumour volume decreased by nearly 50% by 7 weeks in treated mice. Weight loss in untreated animals was prevented by ATP. Histological examination of the excised tumour nodules showed necrosis in the ATP-treated tumours only. The presence of P2Y 1 and P2X 7 receptors, previously proposed as extracellular targets for melanoma treatment with ATP, were demonstrated in the excised specimens by immunohistochemistry. This paper provides further support for the use of ATP as a treatment for melanoma.
Embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells are attractive in vitro models of vascular development, therapeutic angiogenesis, and tissue engineering. However, distinct ESC and iPS cell lines respond differentially to the same microenvironmental factors. Developing improved/optimized differentiation methodologies tailored/applicable in a number of distinct iPS and ESC lines remains a challenge in the field. Currently published methods for deriving endothelial cells (EC) robustly generate high numbers of endothlelial progenitor cells (EPC) within a week, but their maturation to definitive EC is much more difficult, taking up to 2 months and requiring additional purification. Therefore, we set out to examine combinations/levels of putative EC induction factors—utilizing our stage-specific chemically-defined derivation methodology in 4 ESC lines including: kinetics, cell seeding density, matrix signaling, as well as medium treatment with vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). The results indicate that temporal development in both early and late stages is the most significant factor generating the desired cells. The generation of early Flk-1+/KDR+ vascular progenitor cells (VPC) from pluripotent ESC is directed predominantly by high cell seeding density and matrix signaling from fibronectin, while VEGF supplementation was NOT statistically significant in more than one cell line, especially with fibronectin matrix which sequesters autocrine VEGF production by the differentiating stem cells. Although some groups have shown that the GSK3-kinase inhibitor (CHIR) can facilitate EPC fate, it hindered the generation of KDR+ cells in our preoptimized medium formulations. The methods summarized here significantly increased the production of mature vascular endothelial (VE)-cadherin+ EC, with up to 93% and 57% purity from mouse and human ESC, respectively, before VE-cadherin+ EC purification.
Overexpression of Gα13, a member of the G12/13 subfamily of heterotrimeric G proteins, has been implicated in cell transformation and the progression of several cancer types. The mechanism through which wildtype, overexpressed Gα13 drives aberrant growth signaling is not known. G protein α subunits are modified post‐translationally by acylation at the N‐terminus, and this addition of a palmitoyl and/or myristoyl group occurs for all mammalian Gα proteins. Gα13 was previously engineered to mutate two cysteine residues necessary for dual palmitoylation. This modification abrogated signaling to serum response factor by Gα13 in both its wildtype and constitutively activated forms. To determine whether myristoylation restores signaling to non‐acylated wildtype Gα13, the protein was modified to harbor the 6 N‐terminal amino acids from GαT, which is myristoylated but not palmitoylated. Loss of growth signaling by non‐palmitoylated, wildtype G13 was rescued by introduction of this myristoylated sequence. In addition, we used cellular fractionation to assess intracellular distribution of these Gα13 variants. Overexpressed, wildtype Gα13 showed a shift from a membrane associated fraction to a soluble pool, which correlated with a sharp increase in serum response factor signaling. Gα13 lacking any acylation sites localized entirely to the soluble fraction but exhibited no growth signaling. We have also examined the role of an N‐terminal polybasic motif in overexpressed wildtype Gα13 signaling, through amino acid substitutions in this region. To query the nucleotide bound state of the soluble Gα13 pool, we combined cell fractionation with trypsin digestion assays. Surprisingly, overexpressed wildtype Gα13 in the soluble fraction was fully degraded, suggesting lack of GTP binding. These constructs have facilitated co‐precipitation experiments to determine whether change in acylation state affects Gα13 binding to specific target proteins.Support or Funding InformationWe acknowledge support from the North Carolina GlaxoSmithKline Foundation, the C.D. Spangler Foundation, UNC Lineberger Comprehensive Cancer Center, and the UNC‐Asheville Undergraduate Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The G12/13 class of heterotrimeric G proteins is unique in its ability to stimulate cellular transformation via overexpression of the wildtype α subunit, Gα12 or Gα13, absent of activating mutations. We have studied the mechanism of wildtype Gα12/13 signaling to the transcriptional activator Serum Response Factor (SRF), a pathway implicated in multiple cancer types. When overexpressed in cultured human embryonic kidney cells, wildtype Gα13 showed robust stimulation of this transcriptional response. This signaling by Gα13 was blunted by overexpression of G protein β1 and γ2 subunits, suggesting that aberrant signaling by the overexpressed α subunit results from stoichiometric imbalance between the trimeric G protein subunits. Next, using an epitope‐tagged Gα13 to track its subcellular location, we discovered the overexpressed α subunit shifted from a membrane‐associated fraction to a soluble fraction, coincident with its sharp increase in SRF signaling. Overexpression of the β1γ2 dimer caused re‐localization of wildtype Gα13 to the membrane‐associated fraction, coincident with its diminished signaling. Interestingly, our preliminary data utilizing a non‐prenylated γ2 subunit suggest the ability of the βγ dimer to suppress signaling by overexpressed, wildtype Gα13 is independent of βγ association with the cell membrane. We currently are examining effects of this non‐membrane bound dimer on subcellular localization of wildtype Gα13. These findings add to our understanding of the mechanism utilized by overexpressed Gα13 to drive aberrant growth signaling.Support or Funding InformationWe acknowledge funding from the North Carolina Biotechnology Center, NC GlaxoSmithKline and CD Spangler Foundations, UNC Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.