The Accumulation of Cyclin-Dependent Kinase Inhibitor p27Is a Primary Response to Staurosporine and Independent of G1 Cell Cycle Arrest

The staurosporine-induced G1 cell cycle arrest was analyzed in a variety of cell lines which includes human tumor cell lines and oncogene-transformed NIH3T3 cell lines. All the cell lines which were sensitive to staurosporine-induced G1 arrest contained a functional retinoblastoma protein (pRB). However, when pRB-lacking fibroblast cells derived from pRB knockout mice were tested they were also sensitive to G1 arrest by staurosporine, indicating that the inactivation of pRB alone is not sufficient for the abrogation of staurosporine-induced G1 arrest. In searching for a common event caused by staurosporine, the cyclin-dependent kinase (CDK) inhibitor protein p27^kip1but not p21^cip1was found to accumulate after staurosporine treatment in all the cell lines examined. This accumulation occurred regardless of the induction of the G1 arrest. The result indicates that the accumulation of p27^kip1is the cell's primary response to staurosporine and that the capability of staurosporine to induce G1 arrest depends on the integrity of cell cycle regulatory components which are downstream of p27^kip1.     

Pre-B cell colony enhancing factor (PBEF), a cytokine with multiple physiological functions

Pre-B cell colony enhancing factor (PBEF) is regarded as a proinflammatory cytokine. Named for its first discovered function as a pre-B cell colony enhancing factor, it has since been found to have many other functions relating to cell metabolism, inflammation, and immune modulation. It has also been found to have intracellular and extracellular forms, with the two overlapping in function. Most of the intracellular functions of PBEF are due to its role as a nicotinamide phosphoribosyltransferase (Nampt). It has been found in human endothelial cells, where it is able to induce angiogenesis through upregulation of VEGF and VEGFR and secretion of MCP-1. In human umbilical endothelial cells, PBEF increases levels of the protease MMP 2/9. PBEF has also been found in a variety of immune cells other than B cells and has been shown to inhibit apoptosis of macrophages. Extracellular PBEF has been shown to increase inflammatory cytokines, such as TNF-α, IL-1β, IL-16, and TGF-β1, and the chemokine receptor CCR3. PBEF also increases the production of IL-6, TNF-α, and IL-1β in CD14⁺ monocyctes, macrophages, and dendritic cells, enhances the effectiveness of T cells, and is vital to the development of both B and T lymphocytes. The purpose of this review is to summarize the recent advances in PBEF research.     

The Serine/Threonine Phosphatase PP4 Is Required for Pro-B Cell Development through Its Promotion of Immunoglobulin VDJ Recombination

PP4 phosphatase regulates a number of crucial processes but the role of PP4 in B cells has never been reported. We generated B cell-specific pp4 knockout mice and have identified an essential role for PP4 in B cell development. Deficiency of PP4 in B lineage cells leads to a strong reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells. In PP4-deficient pro-B cells, immunoglobulin (Ig) DJ_H recombination is impaired and Ig µ heavy chain expression is greatly decreased. In addition, PP4-deficient pro-B cells show an increase of DNA double-strand breaks at Ig loci. Consistent with their reduced numbers, residual PP4-deficient pre-B cells accumulate in the G1 phase, exhibit excessive DNA damage, and undergo increased apoptosis. Overexpression of transgenic Ig in PP4-deficient mice rescues the defect in B cell development such that the animals have normal numbers of IgM⁺ B cells. Our study therefore reveals a novel function for PP4 in pro-B cell development through its promotion of V_HDJ_H recombination.     

M1 macrophage subtypes activation and adipocyte dysfunction worsen during prolonged consumption of a fructose-rich diet

Fructose-rich diet (FRD) has been associated with obesity development, which is characterized by adipocytes hypertrophy and chronic low-grade inflammation. Interaction of adipocytes and immune cells plays a key role in adipose tissue (AT) alterations in obesity. We assessed the metabolic and immune impairments in AT in a murine obesity model induced by FRD at different periods. Adult Swiss mice were divided into groups of 6 and 10 weeks of fructose (FRD 6wk, FRD 10wk) or water intake (CTR 6wk, CTR 10wk). FRD induced increased in body weight, epidydimal AT mass, and plasmatic and liver Tg, and impaired insulin sensitivity. Also, hypertrophic adipocytes from FRD 6wk-10wk mice showed higher IL-6 when stimulated with LPS and leptin secretion. Several of these alterations worsened in FRD 10wk. Regarding AT inflammation, FRD mice have increased TNFα, IL-6 and IL1β, and decrease in IL-10 and CD206 mRNA levels. Using CD11b, LY6C, CD11c and CD206 as macrophages markers, we identified for first time in AT M1 (M1a: Ly6C^+/−CD11c⁺CD206⁻ and M1b: Ly6C^+/−CD11c⁺CD206⁺) and M2 subtypes (Ly6C^+/−CD11c⁻CD206⁺). M1a phenotype increased from 6 weeks onward, while Ly6C^+/− M1b phenotype increased only after 10 weeks. Finally, co-culture of RAW264.7 (monocytes cell line) and CTR or FRD adipocytes showed that FRD 10wk adipocytes increased IL-6 expression in non- or LPS-stimulated monocytes. Our results showed that AT dysfunction got worse as the period of fructose consumption was longer. Inflammatory macrophage subtypes increased depending on the period of FRD intake, and hypertrophic adipocytes were able to create an environment that favored M1 phenotype in vitro.     

Interleukin-15 expression affects homeostasis and function of B cells through NK cell-derived interferon-γ

Interleukin-15 (IL-15) is a cytokine important for the development, maturation, and function of many cells of the immune system including NK, NKT, γδT, and CD8⁺ T cells. The relationship between IL-15 and B lymphocytes however, is not well characterized and is the focus of our study. Previous in vitro reports have shown that IL-15 increases proliferation of B lymphocytes and increases antibody secretion however, this relationship remains inadequately defined in vivo. The focus of this study was to examine the role of IL-15 in B cell homeostasis and function in vivo using mice that either over express IL-15 (IL-15tg mice) or are deficient in IL-15 (IL-15^−/− mice) production. Here we report significant differences between the B cell populations of IL-15^−/−, C57BL/6, and IL-15tg mice. In fact, increased expression of IL-15 resulted in a significant decrease in the percentage and absolute number of CD19⁺ cells. In vitro B cell co-cultures implicate interferon-gamma (IFN-γ) as the factor responsible for inhibiting B cell proliferation. We also show that IL-15 expression affects B cell function, as B cells from IL-15 transgenic mice produce greater amounts of IgG and IgA than IL-15 knockout mice in vitro. Interestingly, despite significant differences in B cell numbers in these strains, there were no significant differences in total antibody titers in serum and vaginal washes of these mice. Results from our in vivo and in vitro experiments suggest that altered expression of IL-15 affects B cell homeostasis through the induction of NK cell-derived IFN-γ.     

T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis

Background

Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7.

Methods and Results

Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4⁺ and CD8⁺ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4⁺ and CD8⁺ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4⁺ and CD8⁺ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4⁺ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis.

Conclusion

These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.     

Cancer risk from low dose radiation in Ptch1+/− mice with inactive DNA repair systems: Therapeutic implications for medulloblastoma

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1^+/− mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs^-/-/Ptch1^+/− mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation.     

Adaptive immune neuroprotection in G93A-SOD1 amyotrophic lateral sclerosis mice

Background

Innate neuroimmune dysfunction is a pathobiological feature of amyotrophic lateral sclerosis (ALS). However, links, if any, between disease and adaptive immunity are poorly understood. Thus, the role of T cell immunity in disease was investigated in human G93A superoxide dismutase 1 (SOD1) transgenic (Tg) mice and subsequently in ALS patients.

Methods and Findings

Quantitative and qualitative immune deficits in lymphoid cell and T cell function were seen in G93A-SOD1 Tg mice. Spleens of Tg animals showed reductions in size, weight, lymphocyte numbers, and morphological deficits at terminal stages of disease compared to their wild-type (Wt) littermates. Spleen sizes and weights of pre-symptomatic Tg mice were unchanged, but deficits were readily seen in T cell proliferation coincident with increased annexin-V associated apoptosis and necrosis of lymphocytes. These lymphoid deficits paralleled failure of Copolymer-1 (COP-1) immunization to affect longevity. In addition, among CD4⁺ T cells in ALS patients, levels of CD45RA⁺ (naïve) T cells were diminished, while CD45RO⁺ (memory) T cells were increased compared to age-matched caregivers. In attempts to correct mutant SOD1 associated immune deficits, we reconstituted SOD1 Tg mice with unfractionated naïve lymphocytes or anti-CD3 activated CD4⁺CD25⁺ T regulatory cells (Treg) or CD4⁺CD25⁻ T effector cells (Teff) from Wt donor mice. While naive lymphocytes failed to enhance survival, both polyclonal-activated Treg and Teff subsets delayed loss of motor function and extended survival; however, only Treg delayed neurological symptom onset, whereas Teff increased latency between disease onset and entry into late stage.

Conclusions

A profound and progressive immunodeficiency is operative in G93A-SOD1 mice and is linked to T cell dysfunction and the failure to elicit COP-1 neuroprotective immune responses. In preliminary studies T cell deficits were also observed in human ALS. These findings, taken together, suggest caution in ascribing vaccination outcomes when these animal models of human ALS are used for study. Nonetheless, the abilities to improve neurological function and life expectancy in G93A-SOD1 Tg mice by reconstitution with activated T cells do provide opportunities for therapeutic intervention.     

UNC5B receptor deletion exacerbates DSS‐induced colitis in mice by increasing epithelial cell apoptosis

The netrin-1 administration or overexpression is known to protect colon from acute colitis. However, the receptor that mediates netrin-1 protective activities in the colon during colitis remains unknown. We tested the hypothesis that UNC5B receptor is a critical mediator of protective function of netrin-1 in dextran sodium sulfate (DSS)-induced colitis using mice with partial deletion of UNC5B receptor. DSS colitis was performed in mice with partial genetic UNC5B deficiency (UNC5B^+/− mice) or wild-type mice to examine the role of endogenous UNC5B. These studies were supported by in vitro models of DSS-induced apoptosis in human colon epithelial cells. WT mice developed colitis in response to DSS feeding as indicated by reduction in bw, reduction in colon length and increase in colon weight. These changes were exacerbated in heterozygous UNC5B knockout mice treated with DSS. Periodic Acid-Schiff stained section shows damages in colon epithelium and mononuclear cell infiltration in WT mice, which was further increased in UNC5B heterozygous knockout mice. This was associated with large increase in inflammatory mediators such as cytokine and chemokine expression and extensive apoptosis of epithelial cells in heterozygous knockout mice as compared to WT mice. Overexpression of UNC5B human colon epithelial cells suppressed DSS-induced apoptosis and caspase-3 activity. Moreover, DSS induced large amount of netrin-1 and shRNA mediated knockdown of netrin-1 induction exacerbated DSS-induced epithelial cell apoptosis. Our results suggest that UNC5B is a critical mediator of cell survival in response to stress in colon.     

Lipopolysaccharides-Induced Suppression of Innate-Like B Cell Apoptosis Is Enhanced by CpG Oligodeoxynucleotide and Requires Toll-Like Receptors 2 and 4

Innate-like B lymphocytes play an important role in innate immunity in periodontal disease through Toll-like receptor (TLR) signaling. However, it is unknown how innate-like B cell apoptosis is affected by the periodontal infection-associated innate signals. This study is to determine the effects of two major TLR ligands, lipopolysaccharide (LPS) and CpG-oligodeoxynucleotides (CpG-ODN), on innate-like B cell apoptosis. Spleen B cells were isolated from wild type (WT), TLR2 knockout (KO) and TLR4 KO mice and cultured with E. coli LPS alone, P. gingivalis LPS alone, or combined with CpG-ODN for 2 days. B cell apoptosis and expressions of specific apoptosis-related genes were analyzed by flow cytometry and real-time PCR respectively. P. gingivalis LPS, but not E. coli LPS, reduced the percentage of AnnexinV⁺/7-AAD^- cells within IgM^highCD23^lowCD43^-CD93^- marginal zone (MZ) B cell sub-population and IgM^highCD23^lowCD43⁺CD93⁺ innate response activator (IRA) B cell sub-population in WT but not TLR2KO or TLR4KO mice. CpG-ODN combined with P. gingivalis LPS further reduced the percentage of AnnexinV⁺/7-AAD^- cells within MZ B cells and IRA B cells in WT but not TLR2 KO or TLR4 KO mice. Pro-apoptotic CASP4, CASP9 and Dapk1 were significantly down-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT but not TLR2 KO or TLR4 KO mice. Anti-apoptotic IL-10 was significantly up-regulated in P. gingivalis LPS- and CpG-ODN-treated B cells from WT and TLR2 KO but not TLR4 KO mice. These results suggested that both TLR2 and TLR4 signaling are required for P. gingivalis LPS-induced, CpG-ODN-enhanced suppression of innate-like B cell apoptosis.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.     

A Critical Function of Mad2l2 in Primordial Germ Cell Development of Mice

The development of primordial germ cells (PGCs) involves several waves of epigenetic reprogramming. A major step is following specification and involves the transition from the stably suppressive histone modification H3K9me2 to the more flexible, still repressive H3K27me3, while PGCs are arrested in G2 phase of their cycle. The significance and underlying molecular mechanism of this transition were so far unknown. Here, we generated mutant mice for the Mad2l2 (Mad2B, Rev7) gene product, and found that they are infertile in both males and females. We demonstrated that Mad2l2 is essential for PGC, but not somatic development. PGCs were specified normally in Mad2l2^−/− embryos, but became eliminated by apoptosis during the subsequent phase of epigenetic reprogramming. A majority of knockout PGCs failed to arrest in the G2 phase, and did not switch from a H3K9me2 to a H3K27me3 configuration. By the analysis of transfected fibroblasts we found that the interaction of Mad2l2 with the histone methyltransferases G9a and GLP lead to a downregulation of H3K9me2. The inhibitory binding of Mad2l2 to Cyclin dependent kinase 1 (Cdk1) could arrest the cell cycle in the G2 phase, and also allowed another histone methyltransferase, Ezh2, to upregulate H3K27me3. Together, these results demonstrate the potential of Mad2l2 in the regulation of both cell cycle and the epigenetic status. The function of Mad2l2 is essential in PGCs, and thus of high relevance for fertility.     

New Castanospermine Glycoside Analogues Inhibit Breast Cancer Cell Proliferation and Induce Apoptosis without Affecting Normal Cells

sp²-Iminosugar-type castanospermine analogues have been shown to exhibit anti-tumor activity. However, their effects on cell proliferation and apoptosis and the molecular mechanism at play are not fully understood. Here, we investigated the effect of two representatives, namely the pseudo-S- and C-octyl glycoside 2-oxa-3-oxocastanospermine derivatives SO-OCS and CO-OCS, on MCF-7 and MDA-MB-231 breast cancer and MCF-10A mammary normal cell lines. We found that SO-OCS and CO-OCS inhibited breast cancer cell viability in a concentration- and time-dependent manner. This effect is specific to breast cancer cells as both molecules had no impact on normal MCF-10A cell proliferation. Both drugs induced a cell cycle arrest. CO-OCS arrested cell cycle at G1 and G2/M in MCF-7 and MDA-MB-231cells respectively. In MCF-7 cells, the G1 arrest is associated with a reduction of CDK4 (cyclin-dependent kinase 4), cyclin D1 and cyclin E expression, pRb phosphorylation, and an overexpression of p21^Waf1/Cip1. In MDA-MB-231 cells, CO-OCS reduced CDK1 but not cyclin B1 expression. SO-OCS accumulated cells in G2/M in both cell lines and this blockade was accompanied by a decrease of CDK1, but not cyclin B1 expression. Furthermore, both drugs induced apoptosis as demonstrated by the increased percentage of annexin V positive cells and Bax/Bcl-2 ratio. Interestingly, in normal MCF-10A cells the two drugs failed to modify cell proliferation, cell cycle progression, cyclins, or CDKs expression. These results demonstrate that the effect of CO-OCS and SO-OCS is triggered by both cell cycle arrest and apoptosis, suggesting that these castanospermine analogues may constitute potential anti-cancer agents against breast cancer.     

N-acetylcysteine increases the frequency of bone marrow pro-B/pre-B cells, but does not reverse cigarette smoking-induced loss of this subset

Background

We previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC), a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells.

Methodology/Principal Findings

Groups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220⁺CD43⁺) and pre-B (B220⁺CD43⁻) cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21–38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle.

Conclusions/Significance

The failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.     

Rescue of tumor-infiltrating lymphocytes from activation-induced cell death enhances the antitumor CTL response in CD5-deficient mice

The CD5 coreceptor is expressed on all T cells and on the B1a B cell subset. It is associated with TCR and BCR, and modulates intracellular signals initiated by both Ag receptor complexes. Human CD5 contributes to regulation of the antitumor immune response and susceptibility of specific CTL to activation-induced cell death (AICD) triggered by the tumor. In this study, we compared the T cell response to the B16F10 melanoma engrafted into CD5-deficient and wild-type C57BL/6 mice. Compared with wild-type mice, CD5 knockout animals displayed delayed tumor growth, associated with tumor infiltration by T cell populations exhibiting a more activated phenotype and enhanced antitumor effector functions. However, control of tumor progression in CD5(-/-) mice was transient due to increased AICD of CD8(+) tumor-infiltrating T lymphocytes. Remarkably, in vivo protection of T cells from TCR-mediated apoptosis by an adenovirus engineered to produce soluble Fas resulted in a dramatic reduction in tumor growth. Our data suggest that recruitment of tumor-specific T cells in the tumor microenvironment occurs at early stages of cancer development and that tumor-mediated AICD of tumor-infiltrating T lymphocytes is most likely involved in tumor escape from the immune system.     

Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression

Ataxia telangiectasia (AT) is caused by mutational inactivation of the ataxia telangiectasia mutated (Atm) gene, which is involved in DNA repair. Increased oxidative stress has been shown in human AT cells and neuronal tissues of Atm-deficient mice. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme and protects cells against oxidative stress. The purpose of this study is to determine whether ATM induces antioxidant enzyme HO-1 and protects cells from oxidative stress-mediated apoptosis by driving the activation of PKC-δ and NF-κB, by increasing cell viability, and by downregulating DNA fragmentation and apoptotic indicators (apoptosis-inducing factor and cleaved caspase-3). AT fibroblasts stably transfected with human full-length ATM cDNA (YZ5 cells) or the empty vector (MOCK cells) were treated with H₂O₂ as a source of reactive oxygen species (ROS). As a result, transfection with ATM inhibited ROS-induced cell death and DNA fragmentation in MOCK cells. Transfection with ATM induced expression of HO-1 which was mediated by PKC-δ and NF-κB in H₂O₂-treated MOCK cells. ZnPP, an HO-1 inhibitor, and transfection with HO-1 siRNA increased ROS levels and apoptosis, whereas hemin, an HO-1 activator, reduced ROS levels and apoptosis in H₂O₂-treated YZ5 cells. Rottlerin, a PKC-δ inhibitor, inhibited NF-κB activation and HO-1 expression in H₂O₂-treated YZ5 cells. MOCK cells showed increased cell death, DNA fragmentation, and apoptotic indicators compared to YZ5 cells exposed to H₂O₂. In addition, transfection with p65 siRNA increased ROS levels and DNA fragmentation, but decreased HO-1 protein levels in H₂O₂-treated YZ5 cells. In conclusion, ATM induces HO-1 expression via activation of PKC-δ and NF-κB and inhibits oxidative stress-induced apoptosis. A loss of HO-1 induction may explain why AT patients are vulnerable to oxidative stress.