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Retinoid-induced apoptosis and Sp1 cleavage occur independently of transcription and require caspase activation
1Sidney Kimmel Cancer Center, San Diego, California 92121, USA.
Abstract:
Vitamin A and its derivatives, the retinoids, are essential regulators of many important biological functions, including cell growth and differentiation, development, homeostasis, and carcinogenesis. Natural retinoids such as all-trans retinoic acid can induce cell differentiation and inhibit growth of certain cancer cells. We recently identified a novel class of synthetic retinoids with strong anti-cancer cell activities in vitro and in vivo which can induce apoptosis in several cancer cell lines. Using an electrophoretic mobility shift assay, we analyzed the DNA binding activity of several transcription factors in T cells treated with apoptotic retinoids. We found that the DNA binding activity of the general transcription factor Sp1 is lost in retinoid-treated T cells undergoing apoptosis. A truncated Sp1 protein is detected by immunoblot analysis, and cytosolic protein extracts prepared from apoptotic cells contain a protease activity which specifically cleaves purified Sp1 in vitro. This proteolysis of Sp1 can be inhibited by N-ethylmaleimide and iodoacetamide, indicating that a cysteine protease mediates cleavage of Sp1. Furthermore, inhibition of Sp1 cleavage by ZVAD-fmk and ZDEVD-fmk suggests that caspases are directly involved in this event. In fact, caspases 2 and 3 are activated in T cells after treatment with apoptotic retinoids. The peptide inhibitors also blocked retinoid-induced apoptosis, as well as processing of caspases and proteolysis of Sp1 and poly(ADP-ribose) polymerase in intact cells. Degradation of Sp1 occurs early during apoptosis and is therefore likely to have profound effects on the basal transcription status of the cell. Interestingly, retinoid-induced apoptosis does not require de novo mRNA and protein synthesis, suggesting that a novel mechanism of retinoid signaling is involved, triggering cell death in a transcriptional activation-independent, caspase-dependent manner.
Insights
Synthetic retinoids induce cancer cell death by activating caspases, which degrade the transcription factor Sp1. This process triggers apoptosis independently of new protein synthesis, offering a novel anti-cancer mechanism.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Retinoids, vitamin A derivatives, regulate cell growth, differentiation, and carcinogenesis.
- Natural and synthetic retinoids exhibit anti-cancer properties by inducing apoptosis in cancer cells.
Purpose of the Study:
- To investigate the molecular mechanisms underlying retinoid-induced apoptosis in cancer cells.
- To identify key cellular targets and signaling pathways involved in retinoid-mediated cell death.
Main Methods:
- Electrophoretic mobility shift assay (EMSA) to analyze transcription factor DNA binding.
- Immunoblot analysis to detect protein truncation.
- In vitro protease assays using purified Sp1.
- Inhibition studies with protease inhibitors (N-ethylmaleimide, iodoacetamide, ZVAD-fmk, ZDEVD-fmk).
- Caspase activity assays and analysis of caspase substrates.
Main Results:
- Synthetic retinoids induce apoptosis in cancer cell lines.
- Retinoid treatment leads to loss of Sp1 DNA binding activity in T cells.
- A specific protease cleaves Sp1 in apoptotic cells, identified as a cysteine protease involving caspases (caspase 2 and 3).
- Inhibition of Sp1 cleavage and caspases blocks retinoid-induced apoptosis.
- Sp1 degradation occurs early in apoptosis and is independent of de novo protein synthesis.
Conclusions:
- Retinoid-induced apoptosis involves caspase-dependent proteolysis of the transcription factor Sp1.
- Sp1 degradation is an early event in retinoid-induced cell death, impacting basal transcription.
- This pathway represents a novel, transcription-independent mechanism for retinoid signaling in cancer therapy.