Questions about the use of [3H]thymidine incorporation as a reliable method to estimate cell proliferation rate

Murat, J.C.; Gamet, Laurence; Cazenave, Y; Trocheris, Véronique

doi:10.1042/bj2700563b

Cited by 14 publications

(4 citation statements)

References 8 publications

(10 reference statements)

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“…As the use of [3H]-thymidine had previously been subjected to caution (Murat et al 1990) we tried to confirm these results using another experimental protocol. Proliferation was measured on decellularized explants (3-D substrate) where, again, SMC and fibroblasts from young SHR exhibited the proliferation chraracteristics of cells isolated from older rats.…”

Section: Proliferation On Decellularized Explantsmentioning

confidence: 89%

Hyperproliferation of Aortic Smooth Muscle Cells and Fibroblasts From Young SHR Rats Is Not Shared by Endothelial Cells

Battle¹,

Arnal²,

Michel³

1994

Clin Exp Pharma Physio

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1. To study the hypertensive genotypic influence on growth kinetics of the three aortic wall cell types. 2. Using young spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats weighing 160-180 g, we compared the proliferative properties of endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts that were isolated from the thoracic aorta of each strain and cultured. Growth-arrested cells were exposed to P < -thymidine after stimulation with 150 micrograms/mL endothelial cell growth supplement. Proliferation assays were performed by cell seeding on decellularized aortic explants and cell counting 2, 4, 5, 6 and 7 days after seeding. The influence of SMC from SHR on the growth kinetics of EC was evaluated by co-cultures in transwell systems. 3. After stimulation, SMC from SHR exhibited a greater P < -thymidine incorporation rate than those from WKY rats (ratios over controls: 3.90 +/- 0.48 [7] vs 1.85 +/- 0.25 [7] respectively, P < 0.05). This was also true for adventitial SHR fibroblasts: (13.1 +/- 0.6 [6] vs 9.9 +/- 1.0 [6] WKY P < 0.05). On the contrary, there was no difference in the P < -thymidine incorporation rates between EC of each strain, regardless of the passage and the time considered. Cell proliferation on matrix explants confirmed the hyperproliferation of SMC and fibroblasts from SHR, while EC of each strain proliferated equally. Smooth muscle cells from SHR did not influence the growth kinetics of EC in co-culture and vice versa. 4. The changes in growth patterns of aortic cells isolated from young prehypertensive SHR seem to be restricted to SMC and fibroblasts.

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Section: Proliferation On Decellularized Explantsmentioning

confidence: 89%

Hyperproliferation of Aortic Smooth Muscle Cells and Fibroblasts From Young SHR Rats Is Not Shared by Endothelial Cells

Battle¹,

Arnal²,

Michel³

1994

Clin Exp Pharma Physio

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“…After 48 h, to allow for attachment of cells, the medium was changed to serum-free RPMI 1640 containing 0.1% BSA, to remove influences of exogenous steroids and growth factors. The cells were then incubated for a further 24 h to synchronize them into the GO/ GI phase of the cell cycle (Murat et al 1990).…”

Section: Cell Culturementioning

confidence: 99%

NO EFFECT OF KININS ON DNA SYNTHESIS IN LNCaP PROSTATE CANCER CELLS

Reid

Schrader

Morris

1994

Clin Exp Pharma Physio

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1. Prostate has kininogenase activity and expresses members of the tissue kallikrein gene family. The present study examined the effect of exogenous and endogenous kinins on growth of LNCaP prostate adenocarcinoma cells. 2. Rate of DNA synthesis was measured by incorporation over 4 h of [3H]-thymidine into a TCA insoluble fraction of LNCaP cells that had been cultured for 24 h. 3. Increased [3H]-thymidine incorporation was seen in response to 10 nmol/L testosterone (+103 +/- 5 s.e.%), dihydrotestosterone (+113 +/- 14%) and R1881 (+64 +/- 10%) (P < or = 0.001; n = 4). 4. In contrast 0.05, 5 and 1000 nmol/L lysyl-bradykinin had no effect (15 +/- 4, 10 +/- 9 and 5 +/- 3 s.e.%, respectively; n = 7). Des-Arg9[Leu8]-bradykinin (a B1 receptor antagonist) and/or D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (a B2 receptor antagonist), 1 nmol/L, and indomethacin, 5 mumol/L, also had little or no effect. 5. In conclusion, kallidin and endogenous kinins and prostaglandins have little or no effect on DNA synthesis and therefore on the growth of LNCaP cells in comparison to the two-fold stimulation produced by androgens.

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“…Incorporation of tritiated thymidine as a reliable method for estimating cell proliferation has been subject to controversy (Woodcock-Mitchell and Yang, 1982;Dumont et al, 1987;Murat et al, 1991). Therefore, the following experimental conditions were designed to give optimal confidence in the fact that incorporated radioactivity was correlated to DNA synthesis.…”

Section: Measurement Of Dna Synthesismentioning

confidence: 99%

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line

Gamet

Murat

Rémaury

et al. 1992

Journal Cellular Physiology

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Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.

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Questions about the use of [3H]thymidine incorporation as a reliable method to estimate cell proliferation rate

Cited by 14 publications

References 8 publications

Hyperproliferation of Aortic Smooth Muscle Cells and Fibroblasts From Young SHR Rats Is Not Shared by Endothelial Cells

Hyperproliferation of Aortic Smooth Muscle Cells and Fibroblasts From Young SHR Rats Is Not Shared by Endothelial Cells

NO EFFECT OF KININS ON DNA SYNTHESIS IN LNCaP PROSTATE CANCER CELLS

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line

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