Apoptosis and expression of proliferative proteins in the developing nervous system and orofacial region of human embryos

We have studied expression of PCNA and Ki-67 in the developing nervous system, sensory organs and orofacial regions in human embryos and fetuses, using monoclonal antibodies PC-10 and MIB-1 in three-step immunohistochemical method and apoptosis performed by TUNEL technique. Expression of PCNA and Ki-67 increased with the age. Apoptosis was rare in above mentioned regions.     

Silencing of RHEB inhibits cell proliferation and promotes apoptosis in colorectal cancer cells via inhibition of the mTOR signaling pathway

Colorectal cancer (CRC) is commonly known as one of the most prominent reasons for cancer-related death in China. Ras homolog enriched in brain (RHEB) and the mammalian target activity of rapamycin (mTOR) signaling pathway were found correlated with CRC, but their specific interaction in CRC was still to be investigated. Therefore, we explored whether RHEB gene silencing affected the cell proliferation, differentiation, and apoptosis by directly targeting the mTOR signaling pathway in cells previously harvested from CRC patients. A microarray analysis was subsequently conducted to investigate the relationship between RHEB and mTOR. Eighty-three adjacent normal tissues and CRC tissues were selected. Immunohistochemistry was carried out to detect the positive expression rates of RHEB and Ki-67 in the CRC tissues. Cells were then transfected with different siRNAs to investigate the potential effects RHEB would have on CRC progression. The expressions of RHEB, 4EBP1, ribosomal protein S6 kinase (p70S6K), proliferating cell nuclear antigen (PCNA), B cell lymphoma 2 (bcl-2), and bcl-2-associated X protein (bax) were determined and then the cell cycle, cell proliferation, and apoptotic rate were also measured. We identified RHEB and mTOR as upregulated genes in CRC. Cells treated with RHEB silencing showed a decreased extent of mTOR, p70S6K, 4EBP1 phosphorylation and expression of RHEB, Ki-67, mTOR, p70S6K, 4EBP1, bcl-2, and PCNA as well as decreased activity of cell proliferation and differentiation; although, the expression of bax was evidently higher. Collectively, our data propose the idea that RHEB gene silencing might repress cell proliferation and differentiation while accelerating apoptosis via inactivating the mTOR signaling pathway.     

Apoptosis in peripheral neuroblastic tumors. Immunohistochemical expression of bcl-2 and p53 is related to DNA fragmentation

We examined 111 cases of neuroblastoma (NB), searching for how NB relates to apoptotic control and other prognostic factors. Immunohistochemistry using avidin-biotin-peroxidase was carried out for bcl-2 and p53 proteins. Apoptosis was analyzed by in situ detection of chromosomal breakdown. DNA ladders were detected by electrophoresis and amplification of MYCN was carried out by PCR and Southern blot. Statistical analyses were performed with Pearson's chi2 and Kruskal-Wallis tests and Cox's regression. We found expression of bcl-2 protein mainly in cases of neuroblastoma without differentiation and in stages 3 and 4. Expression of p53 protein showed a correlation with bcl-2 and the apoptotic phenomenon; apoptosis was found mainly in favorable cases. Multivariate analysis showed bcl-2 protein expression to be the most independent risk factor. The study of apoptosis could be important for the design of therapies to treat neuroblastoma.     

Selected markers of proliferation and apoptosis in the parathyroid lesions: a spatial visualization and quantification

The aim of the paper was to apply a method for quantitative assessment of proliferation and apoptosis markers, based on their 3D visualization, in cases of parathyroid adenoma and hyperplasia. Material was obtained from 49 patients (32 females and 17 males) with primary hyperparahyroidism. Quantitative immunohistochemistry studies of Ki-67, proliferating cell nuclear antigen (PCNA) and bcl-2 were performed on digital microscopy images with the use of 3D visualization. The use of spatial visualization method allowed us to perform objective quantitative assessment of the studied immunohistochemical markers. The average cell nuclear fraction of Ki67+ was 1.8% in hyperplasia and 1.9% in adenoma cases while 3.5% in the controls. The highest expression of PCNA was found in parathyroid hyperplasia (22.9%) and significantly decreased in adenoma (12.5%) and in the control group (16.8%). The lower expression of bcl-2 in hyperplasia cases (mean area fraction of 0.172 per 1 μm², in contrast to 0.643 in adenomas and 0.648 in control) suggested that principal cells can be ready for apoptosis and may confirm the important role of bcl-2 protein in etiopathogenesis of hyperplasia of the parathyroid gland while PCNA might be a useful marker for differentiating adenoma from early hyperplasia in primary hyperparahyroidism cases.     

Prognostic value of apoptosis in breast cancer (pT1-pT2). A TUNEL, p53, bcl-2, bag-1 and Bax immunohistochemical study

Apoptosis or programmed cell death produces cells breaking into several fragments of nuclei, cytoplasm or both nuclei and cytoplasm, known as apoptotic bodies which can be visualized in haematoxylin-eosin staining. Some genes (promoters and suppressors) control this process and certain mutations may induce the expression of abnormal proteins, which can be detected by immunohistochemical staining. Apoptosis can be detected by the TUNEL method either identifying apoptotic bodies or cells at the initial stages of the fragmentation process. We have studied 186 cases of infiltrating ductal breast carcinoma, stages pT1-pT2, and analysed the prognostic significance of tumour recurrence and overall survival of apoptotic index (AI) through univariate and multivariate analysis. We have also studied the immunohistochemical protein expression of apoptosis promoter and suppressors gene (p53, nuclear expression; bcl-2 and Bax, cytoplasm expression; BAG-1, nuclear and cytoplasm expression). The results indicate prognostic significance of p53 and bcl-2 related to patient death and bcl-2 and tumour size to tumour recurrence, bcl-2 acting as a protector factor (apoptotic suppressor) in both situations. On the other hand, we have not found useful prognostic information of AI either to tumour recurrence or overall survival in univariate or multivariate studies. In this study, Bax expression does not provide a new prognostic role in breast carcinoma, although it contrasts to the bcl-2 action and accelerates death.     

Apical protuberances of supporting cells in the regio olfactoria of the mole,Talpa europaea,Linnaeus, 1758 (Insectivora,Talpidae)

Summary The regio olfactoria of the mole, Talpa europaea, was studied by scanning and transmission electron microscopy. Peculiar structural differentiations, i.e. ovoid-shaped, balloon-like protuberances were found on the surface of the supporting cells. The apical portion of these protuberances contained finely dispersed granular material, whereas in their central part vesicular extensions of the smooth endoplasmic reticulum were observed.     

Involvement of p53 and Bcl-2 family proteins in regulating programmed cell death and proliferation in human embryogenesis

Homeostasis and development in vertebrates are regulated by cell proliferation, differentiation and death. Permeability of mitochondrial membranes, a decisive feature of apoptosis, is regulated by Bcl-2 family regulators. Protein p53 is able to reduce bcl-2 and promote bax expression. This study focused on the immunohistochemical detection of the expression levels of Bcl-2 family regulators (anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bcl-Xs and Bax), p53, and PCNA as a marker of proliferation, together with the evaluation of the level of apoptosis in human embryos (anlage of limbs, axial skeleton, metanephros, and intestine). Expression of observed proteins was assessed by a three-step immunohistochemistry and evidenced by the double-staining technique. Apoptosis was detected by the TUNEL technique. This study provided circumstantial evidence of the exclusive role of Bcl-2 and Bcl-XL proteins in the inhibition of apoptosis - only rarely were the Bcl-2/ Bcl-XL positive cells stained by TUNEL. The role of pro-apoptotic members of Bcl-2 family remains ambiguous, as TUNEL positive cells are both Bax/Bcl-Xs positive and negative. This study provided substantial evidence that expression patterns of observed proteins are neither fully explainable by "rheostat" theory, nor are the findings obtained from animal model tissue or cell culture commonly applicable to human embryos.     

Repercussions of castration and vasectomy on the ductal system of the rat ventral prostate

Diseases, such as cancer and benign prostatic hyperplasia, are related to disruption of the mechanism regulating the balance between cell proliferation and apoptosis in prostatic cells. Since castration and vasectomy might alter that balance, this study evaluates the cell proliferation, apoptosis and height of the secretory epithelium of the ventral-prostate ductal system post-castration and vasectomy. Immunohistochemical (PCNA and Ki67), cytochemical (Fuelgen reaction) and morphometric investigation have been carried out. Cell proliferation indices decreased significantly in both regions of the ventral-prostate ductal system after castration compared to the sham-operated group. The apoptotic index increased significantly after 48 h, declining 7 days post-castration. The cell proliferation indices did not differ after 48 h significantly; however, they increased 7 days post-vasectomy in both regions. The apoptotic index did not differ significantly in either time post-vasectomy. Castration caused an imbalance in favor of apoptosis, whereas vasectomy caused an imbalance in favor of cell proliferation.     

Proliferation and apoptosis in solid tumors. Analysis by laser scanning cytometry

OBJECTIVE: To examine the relationship between apoptosis and proliferation in a series of human solid malignant tumors, making use of objective, reproducible techniques newly developed for laser scanning cytometry (LSC). STUDY DESIGN: Apoptosis was detected by in situ end labeling of DNA strand breaks with FITC-conjugated nucleotide. Proliferation was detected by Ki-67 antibody. Two parameters were detected independently and simultaneously with DNA measurement on aliquots of cell suspensions obtained by mechanical dissociation of fresh tumors and placed on microscope slides. RESULTS: The number of cells undergoing apoptosis varied from 0.5% to 28.1% (average, 5.4 +/- 6.0). Aneuploid tumors showed a higher percentage of apoptotic cells (7.9 +/- 7.2) as compared to diploid tumors (3.4 +/- 4.0). Tumors with the greatest number of apoptotic cells on LSC also had the largest number of apoptotic cells on light microscopic examination. The number of cells labeled by Ki-67 ranged from 1.7% to 56.7% (average, 20.0 +/- 15.5). Aneuploid tumors were characterized by a higher Ki-67 index (average, 28.3 +/- 14.3%) than the diploid tumors (13.2 +/- 13.3%). CONCLUSION: Overall, there was a very weak or no correlation between apoptosis and proliferation. However, a subset of aneuploid tumors had a high percentage of cells positive for Ki-67 and low percentage of apoptotic cells. Diploid tumors did not show any correlation between apoptosis and proliferation, although many of those tumors had both low apoptotic and proliferative indices. Whether those differences are of prognostic significance remains to be determined in follow-up studies that include more cases and clinical data. Here we have shown that LSC is a powerful new tool of potential clinical value for fast, objective analysis of apoptosis, proliferation and DNA ploidy in solid malignant tumors.     

Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland

Apoptosis and proliferation of parenchymal cells during atrophy of rat submandibular gland induced by double duct ligation were investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) and transmission electron microscopy (TEM). At 2 and 3 days after ligation, increased PCNA positive cells and mitoses were seen in ducts; thereafter PCNA positive cells decreased in number. At 3 and 4 days, the acinar cell population rapidly decreased, with many remaining TUNEL positive acinar cells. During this period, TEM showed typical apoptotic acinar cells that were phagocytosed by adjacent acinar cells or intraepithelial macrophages. After 7 days, most acinar cells had disappeared, leaving prominent residual ducts; a few acinar cells remained, especially at the lobule periphery. Submandibular gland duct ligation thus induced marked depletion of acinar cell by apoptosis and a concurrent short-lived cycle of duct cell proliferation.     

The lens controls cell survival in the retina: Evidence from the blind cavefish Astyanax

The lens influences retinal growth and differentiation during vertebrate eye development but the mechanisms are not understood. The role of the lens in retinal growth and development was studied in the teleost Astyanax mexicanus, which has eyed surface-dwelling (surface fish) and blind cave-dwelling (cavefish) forms. A lens and laminated retina initially develop in cavefish embryos, but the lens dies by apoptosis. The cavefish retina is subsequently disorganized, apoptotic cells appear, the photoreceptor layer degenerates, and retinal growth is arrested. We show here by PCNA, BrdU, and TUNEL labeling that cell proliferation continues in the adult cavefish retina but the newly born cells are removed by apoptosis. Surface fish to cavefish lens transplantation, which restores retinal growth and rod cell differentiation, abolished apoptosis in the retina but not in the RPE. Surface fish lens deletion did not cause apoptosis in the surface fish retina or affect RPE differentiation. Neither lens transplantation in cavefish nor lens deletion in surface fish affected retinal cell proliferation. We conclude that the lens acts in concert with another optic component, possibly the RPE, to promote retinal cell survival. Accordingly, deficiency in both optic structures may lead to eye degeneration in cavefish.     

灵芝肽诱导人肝癌HepG2细胞凋亡的研究

研究了灵芝肽(GLP)在体外对人肝癌HepG2细胞凋亡的影响,并初步探讨了其作用机制。结果显示,透射电镜下可见细胞染色质浓缩、聚集于核边缘成块状,形成典型的凋亡小体;GLP使HepG2细胞阻滞于G0/G1期,随着GLP浓度升高,其G0/G1期的细胞比例随之增加;同时细胞的早期、晚期和总的凋亡率亦均随之增加,存在剂量-效应关系;Western blotting检测结果显示,抑制凋亡基因bcl-2和survivin表达下调,而促凋亡基因p53表达上调,并且都存在剂量依赖性;细胞凋亡的关键蛋白酶caspase-3被激活,并且caspase-3酶活性与GLP浓度亦有剂量依赖性。提示GLP体外可诱导人肝癌HepG2细胞凋亡,其作用机制可能与bcl-2和survivin表达下调、p53表达上调及Caspase-3被激活有关。     

Role of apoptosis and mitosis during human eye development

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.     

小鼠生精细胞增殖与凋亡的年龄变化

为研究雄性小鼠睾丸在发生,发育过程生精细胞增殖与凋亡的年龄变化,本研究采用了免疫细胞化学,凋亡细胞原位检测,电镜及体视学图象分析等方法,对胚胎15天到生后10月后发育阶段生精细胞的超微结构,PCNA表达,凋亡情况进行了较深入地研究,结果：（1）精原细胞PCNA反应在胚胎15天为阳性,从胚胎18天到生后5天,降为弱阳性,而阳性的精原细胞在生后7天重新出现,一直到生后6月,仍可见部分精原细胞呈阳性反应;（2）生后3天,可见凋亡的精原细胞染色质浓缩,核膜出现明显的核周隙,核碎裂,凋亡细胞数从生后1天到生后第3周有增加的趋势,于生后第3周出现峰值,之后降低,之后降低,结论：（1）PCNA阳性细胞而密度到生后第2周出现峰值,而凋亡细胞数于生后3周出现峰值,生精细胞凋亡的高峰要滞后其增殖峰1周左右,而且与其所处生精周期的特定阶段有关;（2）精原细胞在胚胎发育过程中向曲细精管周边迁移,其排列由无序到有序;（3）在生后各发育 ,精原细胞始终保持DNA复制的能力。     

Onset and time course of apoptosis in the developing zebrafish retina

In mammalian development, apoptosis spreads over the retina in consecutive waves and induces a remarkable amount of cell loss. No evidence for such consecutive waves has been revealed in the fish retina so far. As the zebrafish is of growing importance as a model for retinal development and for degenerative retinal diseases, we examined the onset and time course of apoptosis in the developing zebrafish retina and in adult fish. We found that apoptosis peaked in the ganglion cell layer (GCL) and inner nuclear layer (INL) in early developmental stages (3-4 days post-fertilization; dpf) followed by a second, but clearly smaller wave at 6-7dpf. Apoptosis in the outer nuclear layer (ONL) started at 5dpf and peaked at 7dpf. This late-onset high peak of apoptosis of photoreceptors is different from that of all other species examined to date. With 1.09% of cells in the GCL and 1.10% in the ONL being apoptotic, the rate of apoptosis in the developing zebrafish retina was conspicuously lower than that observed in other vertebrates (up to 50% in GCL). During development (2-21dpf), apoptotic waves were most obvious in the central retina, whereas in the periphery near the marginal zone (MZ), apoptosis was much lower; in adult animals, practically no apoptosis was present in the central retina but it still occurred near the MZ. Our data show that the onset and time course of apoptosis in the GCL and INL of the zebrafish is comparable with other vertebrates; however, the amount of apoptosis is clearly reduced. Thus, apoptosis in the zebrafish retina may serve more as a mechanism for the fine tuning of the retinal neuronal network after mitotic waves during development or in remaining mitotic areas than as a mechanism for eliminating large numbers of excess cells.     

Adenovirus-mediated p21((WAF1/SDII/CIP1)) gene transfer induces apoptosis of human cervical cancer cell lines

p21((WAF1/SDII/CIP1)) (p21) arrests cell growth by inhibiting cyclin-depend kinases. To explore the potential of using p21 for the gene therapy of cervical cancer, we infected human papillomavirus (HPV)-positive cervical cancer cells (HeLa, SiHa, and Z172) and HPV-negative cervical cancer cells (C33A) with recombinant adenovirus encoding p21 cDNA. The results revealed that effective inhibition of cell growth could be achieved by sense p21 adenovirus but not antisense p21 adenovirus infection and occurred through apoptosis as measured by DNA fragmentation and chromatin condensation. Apoptosis was also observed in xenografts of human cervical cancer cells infected with sense p21 adenovirus, as confirmed by in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). The apoptosis was not prevented by overexpression of the bcl-2 transgene. To sum up, the apoptotic effect suggests that p21 should be a tumoricidal agent instead of a tumoristatic agent in preventing cervical cancers. In addition, our report substantiates the combination of the high efficiency of adenovirus vector-mediated gene delivery and the apoptotic effect of p21.     

Apoptosis resistance in pigmented villonodular synovitis

OBJECTIVE: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. METHODS: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multi-parametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. RESULTS: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p=0.037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expression of both caspase3 (p=0.017) and bcl-2 (p=0.0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found). CONCLUSION: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions.     

Cell proliferation and apoptosis in stromal corneal dystrophies

The aim of our study was to evaluate corneal cell proliferation and apoptosis in cases of granular, macular and lattice dystrophy, and to provide evidence which may help to clarify whether apoptosis is a pathogenic factor in any of these dystrophies. The study group comprised 39 eyes (from 33 patients) which had undergone penetrating keratoplasty (PK) for stromal dystrophies: these comprised 12 eyes (from 9 patients, 55.5% males) with granular dystrophy, 13 eyes (12 patients, 33.3% males) with macular dystrophy, and 14 eyes (13 patients, 61.5% males) with lattice type I dystrophy. A further 4 corneal buttons from enucleated eyes of 4 patients with choroideal melanoma served as controls. Immunocytochemical analysis of Ki67 (DNAcon Kit, DakoCytomation A/S, Glostrup, Denmark) was used for evaluation of cell proliferation. Apoptosis was detected by use of the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling) assay method (Apoptag reagent, Q-Biogene, Strasbourg, France). Statistical comparisons were made using the Mann-Whitney test. No Ki67-positive cells were detected in the study-group or control corneas. In control corneas no apoptotic activity was found. In the study group the mean (normalised) apoptotic keratocyte number was 1.1+/-1.7 in granular dystrophy and 0.5+/-1.1 in lattice type I dystrophy (p = 0.36, 0.63 respectively). Compared to the controls, the difference was statistically significant only for macular dystrophy (1.6+/-1.2; p = 0.01). Keratocyte apoptosis seems to be a concomitant or pathogenic factor in macular dystrophy. However, the pathways that are triggered to result in increased apoptotic cell death remain to be clarified.     

Apoptotic markers for tumor recurrence: a minireview

The control of apoptotic mechanisms is integral to many aspects of tumor biology and appears to be involved in the process of recurrence. Apoptosis serves as an essential mechanism to prevent the proliferation of cells with a higher mutation rate, thus tempering malignant transformation. Most antineoplastic therapies function by triggering apoptosis in sensitive cells. Resistance to treatment may result from specific inhibition of apoptotic signaling. Chemotherapy or radiation may increase the mutation rate and hasten tumor evolution in cancer cells that are resistant to apoptosis. Summarizing the current evidence regarding the usefulness of various apoptotic markers for predicting tumor recurrence, the most extensively studied appear to be bcl-2 and p53, as well as the apoptotic rate itself, with promising prognostic potential in several neoplasias. Investigative results, however, mostly refer to multiple single-center retrospective studies, awaiting validation by large prospective clinical trials. Despite initial optimism, it becomes apparent that the measurement of one or more gene products is inadequate to directly predict a phenomenon as complex as the clinical outcome. One of the challenges that is only beginning to be addressed is the combined assessment of traditional prognostic parameters and molecular biomarkers by creating models or equations to predict the likelihood of recurrence. Such screening of patients may help define prognostic categories and influence treatment decisions.     

bcl-2, a novel reguator of cell death

The bcl-2 gene product, a 25 kDa membrane protein residing at mitochondrial, microsomal and nuclear membrane sites within many cell types, is a broad and potent inhibitor of cell death by apoptosis. A family of bcl-2-related genes with death-inhibiting or -promoting activities has recently been described, indicating a potentially quite complex cell death regulatory network at the level of gene expression and protein-protein interactions. The function of bcl-2 may be to regulate a final common pathway in apoptosis. Current hypotheses suggest that oxidative stress, specific proteolytic activity or cell cycle control may be common elements in apoptosis through which bcl-2 exerts its survival function. Based on the extent to which elements of apoptotic pathways overlap with non-apoptotic cellular functions, the physiological role of bcl-2 may also extend to other cellular processes such as differentiation and proliferation.