Inaccuracies in MTS Assays: Major Distorting Effects of Medium, Serum Albumin, and Fatty Acids

Huang, Kuang Tzu; Chen, Yen Hao; Walker, Ameae M.

doi:10.2144/04373st05

Cited by 80 publications

(69 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The medium was changed daily. At the end of the growth period, the medium was changed to serum-free RPMI 1640 to measure relative viable cell number using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium, inner salt assay; Promega, Madison, WI], as described previously (48). Western blot of the VDR and p21 response to unmodified prolactin or S179D PRL after incubation for 3 days.…”

Section: Methodsmentioning

confidence: 99%

S179D Prolactin Increases Vitamin D Receptor and p21 through Up-regulation of Short 1b Prolactin Receptor in Human Prostate Cancer Cells

Ginsburg

Vonderhaar

et al. 2005

Cancer Research

Self Cite

View full text Add to dashboard Cite

In this study, we further investigated the mechanisms by which pseudophosphorylated prolactin (S179D PRL) inhibits the growth of human prostate cancer cells. When treated with S179D PRL for 3 days, LnCAP cells responded by increasing expression of the vitamin D receptor (VDR) and the cell cycle regulatory molecule, p21, whereas PC3 and DU145 cells did not. After 5 days of treatment, both PC3 and DU145 cells responded. Untreated LnCAP cells express the short 1b form (SF1b) of the human prolactin receptor, but DU145 and PC3 cells express only low amounts of this receptor until elevated by treatment with S179D PRL. DU145 and PC3 cells become sensitive to the negative effects of S179D PRL on cell number after induction of the SF1b. Transfection of either SF1b or SF1a into PC3 or DU145 cells made them sensitive to S179D PRL in the 3-day time frame, a finding that was not duplicated by transfection with the long form of the receptor. Treatment of LnCAP cells with S179D PRL increased long-term activation of extracellular signal-regulated kinase 1/2 (ERK1/2). This did not occur in PC3 and DU145 cells until transfection with SF1a/ SF1b. Blockade of ERK signaling eliminated S179D PRLstimulated expression of the VDR and p21 in LnCAP cells and transfected PC3 and DU145 cells. We conclude that initiation of alternative splicing to produce SF1b, and subsequent altered signaling, contribute to the growth inhibitory mechanisms of S179D PRL. This is the first indication of a role for short prolactin receptors in the regulation of cell proliferation. (Cancer Res 2005; 65(16): 7509-15)

show abstract

Section: Methodsmentioning

confidence: 99%

S179D Prolactin Increases Vitamin D Receptor and p21 through Up-regulation of Short 1b Prolactin Receptor in Human Prostate Cancer Cells

Ginsburg

Vonderhaar

et al. 2005

Cancer Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…After 24-h serum withdrawal, cells were treated with vehicle or hormone for 48 h. Assays were done using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay Kit (MTS; Promega) according to the manufacturer's protocol with modifications as previously described (54).…”

Section: Effects On Cell Numbermentioning

confidence: 99%

Prolactin Does Not Require Insulin-Like Growth Factor Intermediates but Synergizes with Insulin-Like Growth Factor I in Human Breast Cancer Cells

Carver

Schuler²

2008

Molecular Cancer Research

View full text Add to dashboard Cite

Insulin-like growth factor (IGF)-II is a required intermediate for prolactin-induced up-regulation of cyclin D1 and proliferation in normal murine mammary epithelial cells in vivo and in vitro. However, we have recently shown that prolactin can rapidly induce cyclin D1 protein expression and subsequent proliferation in the MCF-7 human breast cancer cell line, suggesting that prolactin actions can be independent of IGFs in breast disease. Here, we investigate the relationship between these factors and show that prolactin up-regulated transcript levels of both IGF-I and IGF-II, but only after increases in cyclin D1 protein were observed. Moreover, prolactin increased cyclin D1 in the presence of the IGF-I receptor neutralizing antibody AIR3. However, on cotreatment, IGF-I and prolactin elicited cooperative phosphorylation of extracellular signal -regulated kinases 1 and 2 and protein kinase B/AKT, but not signal transducer and activator of transcription 5. This interaction extended to increased activation of activating protein-1 enhancer elements, phosphorylation of glycogen synthase kinase 3B, induction of cyclin D1, and ultimately, increased cell number. It also increased invasive behavior, which correlated with elevated matrix metalloproteinase-2 transcript levels. Interestingly, prolactin augmented phosphorylation at Tyr 1135 and Tyr 1136 of IGF-I receptor on cotreatment with IGF-I, although prolactin alone had no effect. Together, these data indicate that strong cooperative cross talk between prolactin and IGF-I augments biological processes associated with neoplastic progression, with implications for therapeutic strategies.

show abstract

“…After 24 h, cells were incubated with either U-PRL or S179D PRL in human endothelial serum-free medium containing bFGF, as before, and epidermal growth factor (EGF) at 10 ng/ml (Sigma) (SFM). Cells were further incubated for 72 h at 37 C. Cell number was determined using a colorimetric assay (Cell Titer; Promega) with the stringent modifications described previously (Huang et al 2004).…”

Section: Cell Proliferation Assaymentioning

confidence: 99%

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone

Ueda

Özerdem

Chen

et al. 2006

Endocr Relat Cancer

Self Cite

View full text Add to dashboard Cite

S179D prolactin (PRL) is an experimentally useful mimic of naturally phosphorylated human prolactin. S179D PRL, but not unmodified PRL, was found to be anti-angiogenic in both the chorioallantoic membrane and corneal assays. Further investigation using human endothelial in vitro models showed reduced cell number, reduced tubule formation in Matrigel, and reduced migration and invasion, as a function of treatment with S179D PRL. Analysis of growth factors in human endothelial cells in response to S179D PRL showed: a decreased expression or release of endogenous PRL, heme-oxygenase-1, basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor and vascular endothelial growth factor; and an increased expression of inhibitors of matrix metalloproteases. S179D PRL also blocked signaling from bFGF in these cells. We conclude that this molecular mimic of a pituitary hormone is a potent anti-angiogenic protein, partly as a result of its ability to reduce utilization of several well-established endothelial autocrine growth loops, partly by its ability to block signaling from bFGF and partly because of its ability to decrease endothelial migration. These findings suggest that circulating levels of phosphorylated PRL may influence the progression of cancer and, furthermore, that S179D PRL may be a useful anti-angiogenic therapeutic.

show abstract

Inaccuracies in MTS Assays: Major Distorting Effects of Medium, Serum Albumin, and Fatty Acids

Cited by 80 publications

References 14 publications

S179D Prolactin Increases Vitamin D Receptor and p21 through Up-regulation of Short 1b Prolactin Receptor in Human Prostate Cancer Cells

S179D Prolactin Increases Vitamin D Receptor and p21 through Up-regulation of Short 1b Prolactin Receptor in Human Prostate Cancer Cells

Prolactin Does Not Require Insulin-Like Growth Factor Intermediates but Synergizes with Insulin-Like Growth Factor I in Human Breast Cancer Cells

A molecular mimic demonstrates that phosphorylated human prolactin is a potent anti-angiogenic hormone

Contact Info

Product

Resources

About