Effects of homocysteine on mesenchymal cell proliferation and differentiation during chondrogenesis on limb development

Abstract

High levels of homocysteine (Hcy) are related to an increased risk of the occurrence of congenital anomalies, including limb defects. However, few evaluations about how toxic levels of Hcy affect limb development have been reported. We investigated whether Hcy can affect the cell cycle proteins and proteins involved in mesenchymal cell differentiation during limb development, in a chicken embryo model. Embryos were treated with 20 µmol d-l Hcy/50 µl saline at embryonic day 2 and analyzed at embryonic day 6. Untreated control embryos received exclusively 50 µl saline solution. To identify cells in proliferation and cell cycle proteins, as well as Pax1/9 and Sox9 proteins, we performed immunolocalization and flow cytometry analyses using the antibodies anti-phosphohistone H3, anti-p53, anti-p21, anti-proliferating cell nuclear antigen, anti-Pax1, anti-Pax9 and anti-Sox9. No significant differences in cell proliferation were observed between Hcy-treated and untreated embryos. We observed a decrease of the proliferating cell nuclear antigen and p21 proteins, both involved in the G1 phase of cell cycle progression. On the other hand, in mesenchymal cells of the limbs, Hcy induces an increase of p53 protein, which can be activated by DNA damage. In cell differentiation, Hcy induced an increase mainly of Pax9 and Sox9 proteins. Our data indicate that the treatment with Hcy changes the mesenchymal cell dynamics during limb development, but does not change the morphology of the cartilage molds. These findings provide information to understand better the cellular basis of the toxicity of Hcy on chondrogenesis during limb development.

Homocysteine influences the human keratocytes cell cycle

“…It may also be suggested, consistently with Bourckhardt et al, that Hcy activity is not necessarily related to the classical P53-mediated cell cycle regulatory pathway. The changes in P53 levels may be involved not necessarily in regulating cell proliferation and DNA damage repair but in a standard program involving pro-apoptotic activity [13]. However, this does not change the fact that Hcy is involved in regulating the cell cycle at the interphase stage, which is reflected at the level of tissue disorders.…”

MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

“…Then, the cells were dissociated using 0.25% trypsin for 15 min at 37°C and added to 5% FBS under agitation. Subsequently, the samples were centrifuged at 640 ×g for 10 min and the supernatant was collected [ 30 ], incubated with primary antibodies rabbit anti-cyclin E IgG (1 : 1000, Santa Cruz Biotechnology, USA), mouse anti-p21 IgG (1 : 1000, Santa Cruz Biotechnology, USA), and mouse anti- β III tubulin IgG (1 : 1000, Promega, USA) for 1 h, and then incubated for 45 min with the secondary antibodies Alexa Fluor 568 anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG (1 : 1000, Life Technologies, USA). Analyses were separately conducted for each antibody in each treatment.…”