Extent Of Cell Apoptosis Research Articles

FTO inhibits oxidative stress by mediating m6A demethylation of Nrf2 to alleviate cerebral ischemia/reperfusion injury.

Current therapies are of limited efficacy in cerebral ischemia/reperfusion (I/R) injury. Based on the important role of oxidative stress in cerebral I/R injury, this study aimed to explore how the N6-adenosine methylation (m6A) demethylase FTO affects oxidative stress. Middle cerebral artery occlusion/reperfusion (MCAO/R)-induced rat model and oxygen and glucose deprivation/re-oxygenation (OGD/R)-induced SH-SY5Y cells were established as in vivo and in vitro model, respectively. The neurological score of rats was measured, and the volume of cerebral infarction was measured by TTC staining. The levels of FTO, nuclear factor-erythroid 2-related factor (Nrf2), and the activity of m6A demethylase FTO were detected. The m6A methylation level of Nrf2 mRNA was detected by MeRIP experiment. Flow cytometry and MTT assay were used to detect apoptosis and proliferation in vitro. TUNEL assay was used to detect apoptosis in brain tissues. FTO and Nrf2 expressions were decreased in the MCAO/R rat brain tissues and OGD/R SH-SY5Y cells, while the m6A methylation level of Nrf2 mRNA was significantly increased. Overexpression of FTO upregulated Nrf2 expression by decreasing the m6A methylation level of Nrf2 mRNA. m6A binding protein YT521-B homology (YTH) domain family protein 2 (YTHDF2) promoted the degradation of Nrf2 by promoting the m6A methylation level of Nrf2 mRNA. Furthermore, SH-SY5Y cell apoptosis was increased and cell viability was decreased after the addition of methyltransferases METTL 3/14, thus blocking FTO to protect SH-SY5Y cells from oxidative stress injury. In vivo, overexpression of FTO decreased the area of cerebral ischemia infarction and the extent of cell apoptosis. In conclusion, FTO increases Nrf2 expression by mediating m6A demethylation of Nrf2 mRNA, thereby inhibiting oxidative stress response and ultimately alleviating cerebral I/R injury.

Scutellarin ameliorates high glucose-induced vascular endothelial cells injury by activating PINK1/Parkin-mediated mitophagy

Ethnopharmacological relevanceScutellarin (Scu) is one of the main active ingredients of Erigeron breviscapus (Vant.) Hand.-Mazz which has been used to treat cardiovascular disease including vascular dysfunction caused by diabetes. Scu also has a protective effect on vascular endothelial cells against hyperglycemia. However, molecular mechanisms underlying this effect are not clear. Aim of the studyThis aim of this study was to investigate the effect of Scu on human umbilical vein endothelial cells (HUVECs) injury induced by high glucose (HG), especially the regulation of PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy. Materials and methodsHUVECs were exposed to HG to induce vascular endothelial cells injury in vitro. Cell viability was assessed by MTT assay. The extent of cell apoptosis was measured by Hoechst staining and flow cytometry. Mitophagy was assayed by fluorescent immunostaining, transmission electron microscope and immunoblot. Besides, virtual docking was conducted to validate the interaction of PINK1 protein and Scu. ResultsWe found that Scu significantly increased cell viability in HG-treated HUVECs. Scu reduces the expression of Bcl-2, Bax and cytochrome C (Cyt.c) to inhibit apoptosis through a mitochondria-dependent pathway. Meanwhile, Scu improved the overload of reactive oxygen species (ROS), superoxide dismutase (SOD) activity and SOD2 protein expression, and reversed the collapse of mitochondrial membrane potential. Besides, Scu increased autophagic flux, improved the expression of microtubule-associated protein 1 light chain 3 Ⅱ (LC3 II), Beclin 1 and autophagy-related gene 5 (Atg 5) and decreased the expression of Sequestosome1/P62 in HG-treated HUVECs. Furthermore, Scu improved the expressions of PINK1, Parkin, and Mitofusin2, which revealed the enhancement of mitophagy. Moreover, the beneficial effects of Scu on HG-induced low expression of Parkin, overproduction of ROS, and over expressions of P62, Cyt.c and Cleaved caspase-3 were weakened by PINK1 gene knockdown. Molecular docking suggested good interaction of Scu and PINK1 protein. ConclusionThese results suggest that Scu may protect vascular endothelial cells against hyperglycemia-induced injury by up-regulating mitophagy via PINK1/Parkin signal pathway.

Applications of daunorubicin-loaded PLGA-PLL-PEG-Tf nanoparticles in hematologic malignancies: an in vitro and in vivo evaluation.

BackgroundWith the development of drug delivery, novel tools and technological approaches have captured the attention of researchers in recent years. Several target drug delivery systems (DDSs) including nanoparticles (NPs) have been developed as an important strategy to deliver classical medicine.ObjectiveThe objective of this study was to evaluate the application of novel daunorubicin (DNR)-loaded poly(lactic-co-glycolic acid)-poly-l-lysine-polyethylene glycol-transferrin (Tf) nanoparticles (DNR-loaded NPs) in hematologic malignancies in vitro and in vivo.Materials and methodsDNR-loaded NPs were prepared by the modified double-emulsion solvent evaporation/diffusion method, and its microscopic form was observed under scanning electron microscope. Intracellular distribution of DNR was directly detected by fluorescence microscopy. After establishment of a tumor xenograft model by injecting K562 cells into the left leg of nude mice, the therapeutic effect of the DNR-loaded NPs on the growth of tumors was measured by calculating the tumor size, and the relative expression of Caspase-3 protein was detected by immunohistochemical staining. Furthermore, intracellular concentration of DNR and the extent of cell apoptosis in primary leukemia cells were quantified by flow cytometry.ResultsDNR-loaded NPs had a spherical shape of about 180 nm in diameter. DNR-loaded NP group showed a significant enhancement of cellular uptake in K562 cells compared with DNR group. Tumor inhibition rate was higher in DNR-loaded NP group in comparison with DNR group, and the relative expression of Caspase-3 protein was upregulated in DNR-loaded NP group compared with DNR group. Furthermore, DNR-loaded NPs obviously increased intracellular concentration of DNR in primary leukemia cells compared with DNR group, but there was no significant difference in primary cell apoptosis between the two groups. These findings suggest that the novel NP DDS can enhance the performance of conventional antitumor drugs and may be suitable for further application in the treatment of hematologic malignancies.

Non‑covalent proteasome inhibitor PI‑1840 induces apoptosis and autophagy in osteosarcoma cells.

Osteosarcoma (OS) is the predominant form of primary bone malignancy in children and adolescents. Although the combination of chemotherapy and modified surgical therapy leads to marked improvements in the survival rate, the therapeutic outcomes remain unsatisfactory. Therefore, the identification of novel drugs with higher efficacy and fewer side-effects is urgently required. Proteasome inhibitors have been approved by the Food and Drug Administration (FDA) for the treatment of certain cancers, although none of them are directed against OS. Non-covalent proteasome inhibitors, such as PI-1840, are superior to covalent ones in numerous respects in view of their chemical structure; however, to date, no studies have been published on the effects of non-covalent proteasome inhibitors on OS cells. In the present study, the antineoplastic effects of PI-1840 were systematically evaluated in the OS cell lines, MG-63 and U2-OS. Cell viability and morphological changes were assessed by Cell Counting Kit-8 (CCK-8) and live/dead assays. The cell cycle was analyzed using flow cytometry (FCM) and western blot analysis (assessing the levels of the proteins p21, p27, and the tyrosine kinase, WEE1). The extent of cell apoptosis and autophagy were assessed by FCM, western blot analysis [of the apoptosis-associated proteins, microtubule-associated protein 1 light chain 3 α (LC3) and Beclin1], and mRFP-GFP-LC3 adenovirus transfection assay. Transwell and wound healing assays, and western blot analysis of the matrix metalloproteinases (MMPs)2 and 9 were performed to preliminarily evaluate the migration and invasion capability of the cells. In the present study, our results revealed that PI-1840 inhibited the proliferation of OS cells and induced apoptosis, partly due to attenuation of the nuclear factor-κB (NF-κB) pathway. In addition, PI-1840-induced autophagy was detected, and inhibiting the autophagy of the OS cells led to an increase in the survival rate of the U2-OS cells rather than of the MG-63 cells. Furthermore, PI-1840 attenuated the migration and invasion capabilities of the OS cells. In conclusion, the present study revealed PI-1840 to be a promising drug for the treatment of OS.

Long intergenic noncoding RNA-p21 inhibits apoptosis by decreasing PUMA expression in non-small cell lung cancer.

ObjectiveLong noncoding RNAs (lncRNAs) are important mediators in tumor progression. Long intergenic noncoding RNA-p21 (lincRNA-p21) participates in multiple biological processes. This study explored the role of lincRNA-p21 in human non-small cell lung cancer (NSCLC) progression and potential regulatory mechanisms.MethodsLincRNA-p21 expression in NSCLC tissues and cell lines (A549, H1299, H1650, and NCI-H2087) was determined by quantitative real-time PCR. LincRNA-p21 overexpressing and sh-lincRNA-p21 lentiviral were respectively transfected into H1299 and A549 cells. Flow cytometry was used to measure apoptosis. Microarray analysis and RNA pull-down assay were used to predict the target genes of lincRNA-p21. Finally, PUMA siRNA and overexpressing PUMA were transfected into NSCLC cells, and the extent of cell apoptosis was measured. The protein expression levels of the relative genes were confirmed by western blot analysis.ResultsLincRNA-p21 was significantly upregulated in NSCLC tissues and cells. The upregulation of lincRNA-p21 considerably inhibited cell apoptosis while the downregulation of lincRNA-p21 showed the opposite effect. PUMA was a direct target gene of lincRNA-p21 and was negatively correlated with lincRNA-p21 in NSCLC specimens. The anti-apoptotic effect of lincRNA-p21 can be effectively attenuated by the upregulation of PUMA.ConclusionLincRNA-p21 is aberrantly upregulated in NSCLC and inhibits cell apoptosis by decreasing PUMA expression.

Erlotinib induces the human non-small-cell lung cancer cells apoptosis via activating ROS-dependent JNK pathways.

Although erlotinib (ERL) has drawn more and more attention toward its anticancer properties effect, the underlying mechanisms of ERL's anticancer properties effect remain unclear yet. So, the aim of this research was to explore the underlying anticancer mechanisms of ERL and to explore whether the reactive oxygen species (ROS)‐dependent c‐Jun N‐terminal kinase (JNK) pathway contributed to the anticancer properties provided by ERL. In our study, we used MTT assay to detect the anticell growth ability of ERL on human non–small‐cell lung cancer cell lines (A549). The extent of cell apoptosis was determined by Hoechst 33342 staining and fluorescence‐activated cell sorter (FACS) assay. Then, DCFH‐DA and JC‐1 staining were used to monitor intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Finally, the effect of ERL on phosphorylation state of JNK protein and downstream apoptosis concerned proteins were detected by western blotting assay. Results showed that ERL significantly suppressed the growth and reproduction of A549 cells with the concentration rising up in vitro. Hoechst 33342 staining and FACS assay also confirmed the proapoptosis effect of ERL on A549 cells with the concentration rising up. Furthermore, exposure of A549 cells to ERL increased the intracellular ROS production. As expected, intracellular ROS activated the proapoptotic JNK signaling pathway and inhibited the activation of EFGR signaling pathway. Our results also revealed that ERL could induce cell‐cycle arrest at G0/G1 period. Activation of JNK protein decreased MMP and downregulated content of antiapoptotic protein Bcl‐2 concomitant with the upregulated content of proapoptotic protein Bax in A549 cells. In addition, c‐Jun and cleaved caspase‐3 were also activated by the phosphorylated JNK induced by ERL. All of these proapoptosis effect of ERL was reversed by administration of N‐acetylcysteine (NAC), which performed as a ROS scavenger. Our results suggest that ERL induces A549 cells apoptosis via activating ROS‐dependent JNK pathways in human non–small lung cancer cells that provide a new experimental foundation for cancer therapy.

Decreased Vascular Endothelial Growth Factor Expression Is Associated With Cell Apoptosis in Low-Dose Aspirin-Induced Gastric Mucosal Injury

BackgroundThe use of low-dose aspirin (LDA) has emerged as an important cause of gastrointestinal ulcers. The aim of this study was to investigate the association between LDA-induced gastric mucosal injury and the expression of vascular endothelial growth factor (vEGF) and cell apoptosis in elderly Chinese patients. MethodsA total of 136 patients aged 60 to 80 years with LDA-induced (100 mg/d for at least 1 month) gastric mucosal injury and 48 age-matched healthy subjects were enrolled in this study. The patients were divided into a low-severity group and a high-severity group based on their modified Lanza scale scores. Biopsy specimens of gastric mucosa from all participants were subjected to immunohistochemical staining for VEGF expression and terminal deoxynucleotidyl transferase dUTP nick end labeling staining for cell apoptosis. Staining indices and apoptotic indices were applied to assess VEGF expression level and the extent of cell apoptosis. ResultsVEGF expression decreased significantly in the 2 patient groups, whereas the extent of cell apoptosis significantly increased compared with the control group. Furthermore, Spearman's correlation coefficients suggest that VEGF expression levels and the extent of cell apoptosis in gastric mucosae shared a significant correlation with the severity of LDA-induced gastric mucosal injury. Receiver operating characteristics analysis further confirmed these results. Conclusionsour findings provide important clues as to the underlying molecular mechanism behind gastric mucosal injury resulting from exposure to LDA in elderly adults, and also suggest that interventions specifically targeting the pathways associated with angiogenesis and apoptosis may help facilitate the healing process.

Zoledronic acid exerts antitumor effects in NB4 acute promyelocytic leukemia cells by inducing apoptosis and S phase arrest

The aim of this study was to investigate the antitumor effect of zoledronic acid (ZOL) in the NB4 human acute promyelocytic leukemia (APL) cell line and explore the potential mechanism of action of this compound. NB4 cells were exposed to various concentrations (0–200μM) of ZOL. Cell viability was measured by MTS assay. The extent of cell apoptosis and distribution of cells in the different phases of the cell cycle were analyzed with flow cytometry. The expression of apoptosis- and cell cycle-related proteins was assayed by Western blot. The combined effect of ZOL and arsenic trioxide (ATO) on the proliferation of NB4 cells was also determined. The results of this study indicate that ZOL inhibits cell proliferation in a time- and dose-dependent fashion and also induces apoptosis and S phase arrest in a dose-dependent manner. The Western blot analysis confirmed the induction of apoptosis and S phase arrest, revealing that the pro-apoptosis proteins Bax, Puma and activated caspase-9 were upregulated and the anti-apoptosis proteins Bcl-2 and Bcl-xL were downregulated. ZOL at a concentration of 50μM synergized with 0.5μM ATO on the growth inhibition of NB4 cells. Taken together, our data indicate that ZOL exerts a potent antitumor effect on NB4 cells by inducing apoptosis and cell cycle arrest, and that ZOL can synergize with the traditional chemotherapy drug ATO.

Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells

Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

Bim Links ER Stress and Apoptosis in Cells Expressing Mutant SOD1 Associated with Amyotrophic Lateral Sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.

Apoptosis-inducing activity of compounds screened and characterized from cinobufacini by bioassay-guided isolation

Cinobufacini (huachansu), an aqueous extract from the skin of the toad Bufo bufo gargarizans Cantor, is a traditional Chinese medicinal preparation widely used in clinical cancer therapy in China. Here, we screened and identified active compounds of cinobufacini and investigated their apoptosis-inducing effect on HepG2 cells. Screening was performed using bioassay-guided isolation. The effects of different fractions on the proliferation of HepG2 cells were detected by the MTT assay. The extraction and isolation of active fractions were performed by chloroform extraction, silica column chromatography, preparative thin-layer chromatography and high-performance liquid chromatography. Nuclear magnetic resonance (NMR) imaging and electron ionization-mass spectrometry (EI-MS) were used to identify the structure of the active compounds. The extent of cell apoptosis was detected by Hoechst 33258 staining and flow cytometric analysis. Western blot analysis was used to detect the expression of the apoptosis-related proteins Bax and Bcl-2. Through bioassay-guided isolation, two compounds were isolated from cinobufacini. NMR and EI-MS data revealed these compounds to be resibufogenin and cinobufagin. Cinobufagin was determined to be the more efficient of the two in inhibiting the proliferation of HepG2 cells. Hoechst 33258 staining and flow cytometric analysis indicated that cinobufagin induced marked changes in apoptotic morphology and significantly increased the proportion of apoptotic cells in HepG2 cells. Western blot analysis showed that cinobufagin up-regulated Bax expression and down-regulated Bcl-2 expression. In conclusion, we screened and identified two anti-proliferation compounds of cinibufacini, resibufogenin and cinobufagin. The most effective compound, cinobufagin, inhibited cell proliferation by inducing the apoptosis of HepG2 cells. This was potentially triggered by regulation of the Bax/ Bcl-2 ratio.

Bcl-2 Localized at the Nuclear Compartment Induces Apoptosis after Transient Overexpression

Bcl-2 is the best characterized member of a large family of proteins that regulate apoptosis. Although it is established that Bcl-2 localized at the mitochondria functions as an anti-apoptotic protein, the function of Bcl-2 at the nucleus remains unclear. Recently we showed that nuclear compartment-associated Bcl-2 inhibits transcription factor activation. Based on this observation, we hypothesized that presence of Bcl-2 at the nucleus may induce rather than protect cells from apoptosis. Here we investigated the putative apoptotic role of nuclear compartment-associated Bcl-2. Additionally, we examined the role of the Bcl-2 BH4 domain in mediating binding to FKBP38, the Bcl-2 mitochondrial chaperone. Our results demonstrate a novel, pro-apoptotic function for nuclear Bcl-2 and identify the Bcl-2 BH4 domain as a key regulator in mediating Bcl-2/FKBP38 binding. These results indicate that Bcl-2 has a dual role as both a protector and a killer and that the ability to switch roles depends on Bcl-2 subcellular localization.

Photodynamic Therapy Agent with a Built-In Apoptosis Sensor for Evaluating Its Own Therapeutic Outcome in Situ

Identifying the extent of apoptosis in cells or tissues after cancer therapy in real time would be a powerful firsthand tool for assessing therapeutic outcome. We combined therapeutic and imaging functions in one agent, choosing photodynamic therapy (PDT) as an appropriate cancer treatment modality. This agent induces photodamage in irradiated cells and simultaneously identifies apoptotic cells by near-infrared fluorescence. This photodynamic therapy agent with a built-in apoptosis sensor (PDT-BIAS) contains a fluorescent photosensitizer used as an anticancer drug, connected to a fluorescence quencher by a caspase-3 cleavable peptide linker. We demonstrated that cleavage of the peptide linker by caspase-3, one of the executioner caspases involved in apoptosis, results in a detectable increase of fluorescence in solution and in cancer cells after PDT treatment. The apoptosis involvement and drug effectiveness were confirmed by Apoptag and cell viability (MTT) assays supporting the ability of PDT-BIAS to induce and image apoptosis in situ.

Hepatocyte growth factor, transforming growth factor alpha, and their receptors as combined markers of prognosis in hepatocellular carcinoma.

A change in the balance between proliferation and apoptosis in the course of hepatocellular carcinoma (HCC) development and progression has been suspected. We wanted to identify related genes whose mRNA levels could provide markers of severity and prognosis after resection. The extent of cell apoptosis, proliferation, and differentiation was measured with a terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick-end labeling assay, and the Ki-67 index was determined in paired tumor and cirrhotic tissue samples from patients who had undergone HCC resection after diagnosis of hepatitis C-related or alcoholism-related cirrhosis. These patients included two groups with highly versus poorly differentiated tumor cells, and the latter was split into two subgroups of those with versus without early recurrence. The mRNA levels for various apoptosis-related or proliferation-related genes and those for the growth factor/receptor systems were measured by quantitative reverse transcriptase-polymerase chain reaction in paired tumor and cirrhotic liver samples from every patient, and some of the corresponding proteins were detected by immunohistochemistry. In all instances, protein expression was highly heterogeneous within groups and similar between groups. In contrast, some differences in mRNA level between tumor and cirrhotic tissues were quite informative. Low levels of hepatocyte growth factor and transforming growth factor alpha mRNAs were found concomitantly in highly differentiated tumors, whereas overexpression of mRNAs for the cognate receptors c-met and epidermal growth factor receptor were found in poorly differentiated tumors and primarily in patients with early tumor recurrence. These results argue for growth factor-dependent HCC development and provide novel and combined prognosis markers after HCC surgery.

Flow cytometric analysis of p53-induced apoptosis.

The p53 protein is a key player in the cellular response to stress conditions. Activation of p53 induces growth inhibition in the form of cell growth arrest or apoptosis. The latter plays an important role in the tumor suppression function of p53. It is therefore of great interest to understand in detail the mechanisms by which p53 induces apoptosis. In this chapter, we describe a flow cytometric assay for the measurement of p53 apoptotic activity. This assay is applicable for exogenously expressed p53 by transfection as well as for endogenous p53. The p53 protein is detected by intra-cellular fluorescent staining of p53 or by tagging p53 with GFP. The extent of apoptosis in cells expressing p53 is determined by cell cycle distribution using flow cytometry. The protocol described here can be employed to study the regulation of p53-mediated apoptosis and can be broadly applied to other apoptotic proteins.

Apoptosis and expression of related proteins in mammary gland of heifers during early lactation.

Lactation is biologically significant and economically important and it has particular nutritional implications in the case of dairy cows. Programmed cell death is a physiological event during mammary gland involution and also plays an important role in the regulation of milk production and persistence of lactation (Capuco and Akers, 1999), although it is numerically less relevant (4.8% vs 2.4%; Wilde, personal communication; Quarrie et al., 1995). Proteins belonging to the bcl-2 family, namely bax and bcl-2, are involved in the regulation of cell apoptosis (Reed, 1997) which also occurs in the mammary glands of lactating mice (Heermeier et al., 1996) and sheep (Colitti et al., 1999); bax acts as a pro-apoptotic and bcl-2 as an anti-apoptotic agent. The aim of the current research was to investigate the morphological organization and the extent of cell apoptosis and proliferation of mammary cells in heifers during early lactation. The possible relationship between mRNA expression of bax and bcl-2 and cell fate was also studied.