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Updated: Jul 13, 2026
Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death
Published on: December 27, 2016
N-methyl-N'-nitro-N-nitrosoguanidine activates multiple cell death mechanisms in human fibroblasts
Michael W Lee1, Wan-Ju Kim, Dillon I Beardsley
1Department of Biochemistry and Molecular Biology and the UF-Shands Cancer Center Program in Cancer Genetics, Epigenetics and Tumor Virology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Abstract:
Response to genotoxic stress may trigger the activation of distinct mechanisms that serve to promote cell death, including apoptosis and necrosis. In this study we examined the response of human fibroblasts, either proficient or deficient for the damage-activated protein kinase ataxia telangiectasia-mutated (ATM), to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Analysis of both long- and short-term viability shows that both ATM-proficient YZ-5 and ATM-deficient EBS-7 fibroblasts display a cytotoxic response to MNNG. Consistent with activation of apoptosis in response to MNNG, we observed increased caspase-3 cleavage and activity, appearance of fragmented nuclei, and increased staining with annexin V in both ATM-proficient and -deficient fibroblasts. Flow cytometry demonstrated that these cell lines also display a nonapoptotic cell death in response to MNNG. This form of cell death is associated with activation of poly-ADP ribose polymerase (PARP), and analysis of PARP activity indicated increased protein poly(ADP-ribosylation) in YZ-5 when compared to EBS-7. This PARP activity was accompanied by apoptosis-inducing factor release and translocation from the mitochondria to the nucleus. Finally, the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) or the caspase-3 inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone dramatically diminished the cytotoxic response to MNNG, reinforcing the roles for apoptotic and nonapoptotic cell death in human fibroblasts treated with MNNG. From these findings, we conclude that MNNG induces a heterogeneous death response in human fibroblasts.
Insights
Genotoxic stress from MNNG triggers both apoptosis and non-apoptotic cell death in human fibroblasts, regardless of ATM kinase status. Inhibiting PARP or caspase-3 reduces this cytotoxic response.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- Genotoxic stress can induce distinct cell death pathways, including apoptosis and necrosis.
- The protein kinase ataxia telangiectasia-mutated (ATM) plays a role in DNA damage response.
- Human fibroblasts are a model system for studying cellular responses to genotoxic agents.
Purpose of the Study:
- To investigate the cytotoxic response of human fibroblasts, both ATM-proficient and ATM-deficient, to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
- To elucidate the specific cell death mechanisms activated by MNNG, focusing on apoptosis and non-apoptotic pathways.
Main Methods:
- Treatment of ATM-proficient (YZ-5) and ATM-deficient (EBS-7) human fibroblasts with MNNG.
- Assessment of cell viability using long- and short-term assays.
- Analysis of apoptosis markers: caspase-3 cleavage, nuclear fragmentation, and annexin V staining.
- Flow cytometry to detect non-apoptotic cell death.
- Measurement of poly-ADP ribose polymerase (PARP) activity and poly(ADP-ribosylation).
- Monitoring of apoptosis-inducing factor (AIF) release and translocation.
- Inhibition of PARP (using DPQ) and caspase-3 (using benzyloxycarbonyl-VAD-fluoromethyl ketone) to assess their roles in MNNG-induced cytotoxicity.
Main Results:
- Both ATM-proficient and ATM-deficient fibroblasts exhibited a cytotoxic response to MNNG.
- MNNG treatment induced apoptosis, evidenced by increased caspase-3 activity, nuclear fragmentation, and annexin V staining in both cell lines.
- A non-apoptotic cell death pathway was also activated, characterized by PARP activation and increased poly(ADP-ribosylation), particularly in ATM-proficient cells.
- PARP activation was linked to apoptosis-inducing factor release from mitochondria to the nucleus.
- Inhibition of PARP or caspase-3 significantly reduced the MNNG-induced cytotoxic effect.
Conclusions:
- MNNG induces a heterogeneous cell death response in human fibroblasts, involving both apoptosis and a non-apoptotic pathway.
- The ATM kinase status does not abolish the induction of these death pathways by MNNG.
- Both PARP and caspase-3 play critical roles in mediating MNNG-induced cytotoxicity in human fibroblasts.
