In SKOV handled cells, apoptosis induction was weak, as proven by

In SKOV treated cells, apoptosis induction was weak, as shown by DAPI examination , as well as delay before reappearance of proliferating cells was particularly shortened, recurrence occurring just after to days . Right after this time, the DNA written content histogram was identical to that of SKOV cells before exposure . Exposure to g ml cisplatin Publicity of IGROV and OAW cells to C induced a strong blockade in G G phases and massive apoptosis just after and h. In particular, no a lot more OAW adherent cells were observable after h . In the two situations, no recurrence occurred, the cell population becoming entirely eradicated. In contrast, when exposed to C, both resistant cell lines had been able to progress by the cell cycle. IGROV R cells accumulated in G M phases at h, whereas SKOV cells did not during the first two days , reaching G M phases only a few days later on . The majority of IGROV R cells underwent apoptosis following h , obtaining or not endoreplicated their DNA, but a slight proportion of them remained in a position to re start off a fresh cell cycle and also to re colonize the culture flask in about to weeks. From the case of SKOV cells, apoptosis remained a marginal event and cells recovered a standard development pattern immediately after about two weeks.
Bcl xL S expression in ovarian carcinoma cell lines and tumor samples Questioning with regards to the function of Bcl xL S from the sensitivity of ovarian carcinoma cells to cisplatin, we very first studied the basal level of Bcl xL S expression in our cell lines and in the panel of ovarian tumor samples. The two bcl xL and bcl xS mRNAs have been observable by RT PCR in all of the cell lines, even though the level of bcl xs mRNA remained noticeably reduce than that selleck new proton pump inhibitor of bcl xL . Western blot evaluation allowed the detection of Bcl xL protein in the many cell lines, whereas Bcl xS protein remained undetectable . Cytological observation after immunodetection confirmed that Bcl xL was expressed in every single cell line, the observed staining becoming evocative of the mitochondrial localization, as selleckchem inhibitor expected . We also investigated Bcl xL S expression inside a panel of ovarian tumor samples. As within the cell lines, RT PCR analysis showed that each bcl xL and bcl xS mRNAs were expressed in the subset of these tumors, the level of bcl xs mRNA becoming also noticeably reduced than that of bcl xL .
Only the antiapoptotic lengthy form of Bcl x could possibly be detected when western blot examination was carried out . Immunohistochemistry research exposed that of the ovarian PF-04217903 tumors expressed Bcl xL, which has a cytoplasmic localization . bcl xL mRNA expression right after cisplatin publicity As demonstrated by Ribonuclease Protection Assay, bclxL mRNA was very expressed in ovarian tumor cell lines, as when compared to other members of bcl family members .

Handle of apoptosis by Slug and msx in neural crest cells of enti

Control of apoptosis by Slug and msx in neural crest cells of full embryos We analyzed the purpose that Slug and msx play on neural crest apoptosis inside the total embryo. One blastomere of two cell stage embryos was injected with inducible varieties of Slug, msx or their dominant negative constructs . To overcome the early effects of Slug and msx in mesoderm and neural crest induction , the inducible fusion constructs weren’t activated by dexamethasone treatment method right up until stage , and, subsequently, cell death was analyzed by TUNEL. Attributable to the variation in TUNEL staining observed between distinct embryos, we always analyzed each the injected and uninjected side of your similar embryo thoroughly. The ordinary patterning of apoptosis was inhibited inside the injected side from the expression of Slug , when the injection from the Slug dominant damaging only made a reasonable grow during the proportion of apoptotic nuclei . Similarly, apoptosis was strongly inhibited over the injected side following expressing a dominant damaging construct of msx .
As msx is actually a downstream target of BMP, we also analyzed the effect of expressing a dominant adverse kind of the BMP receptor and observed that it strongly inhibited apoptosis over the injected side TW-37 . As it has become described that inhibition of msx can suppress Slug expression when it is actually activated with the late gastrula stage , we analyzed if a comparable relation was taking place concerning these two factors in the mid neurula stage. Embryos injected with the one cell stage together with the inducible constructs in the dominant adverse of msx or Slug, were induced at stage along with the expression of Slug or msx was analyzed, respectively. No result from the expression of Slug was observed when msx dominant negative was expressed , and no result on msx was observed when Slug was expressed ; consequently, when any of those factors are activated following neural crest specification , no mutual control of transcription consider spot. In conclusion, these success indicate that Slug can indeed act as an antiapoptotic factor although msx promotes apoptosis from the neural crest of Xenopus embryos.
An interaction of Slug and msx with all the Bcl Bax proteins is required to control apoptosis Bcl and Bax proteins kind part in the core apoptotic machinery, which can be conserved across species as various as C. elegans and mammals. With the practical degree, Bcl inhibits apoptosis whereas Bax promotes it, whilst the ultimate determination of the cell to execute the program of cell death depends PF-562271 over the stability between the many proteins within the apoptotic machinery. Given that Slug and msx are involved in controlling apoptosis, we chose to analyze the interaction among each one of these factors in isolated animal caps and in total embryos. We injected mRNA encoding Bax at the one particular cell stage, animal caps have been dissected, cultured in vitro, and TUNEL staining was analyzed.

Alk SM did not ectopically activate Smad signaling from the absen

Alk SM didn’t ectopically activate Smad signaling during the absence of activin, indicating that, at these doses, the mutant receptor will not non particularly or constitutively activate the Nodal pathway to an appreciable degree in animal caps. Similarly, Alk SM but not Alk WT rescued endogenous p Smad in SB treated zebrafish embryos at epiboly . Alk SM rescues phenotypic and gene expression defects in SB handled zebrafish embryos Considering Alk SM could restore p Smad signaling in embryos, we employed it to request no matter whether the SB induced phenotype of embryos was likewise unique. Alk SM did indeed rescue the SB phenotype in zebrafish. of embryos injected with pg Alk SM mRNA and subsequently taken care of with M SB displayed phenotypically ordinary head growth . Rescued embryos had two distinct and separate eyes and regular midline structures . The unrescued fraction of embryos quite possibly reflects bad or incomplete dispersal from the injected mRNA throughout the entire embryo. In contrast to Alk SM, wild sort Alk was not able to rescue inhibitor induced head defects .
Embryos injected with mRNA encoding either wild kind or mutant receptors and treated with DMSO had been phenotypically indistinguishable from uninjected embryos, indicating the receptors alone never considerably impact phenotype . A lower dose of mRNA was still in a position to rescue, but was much less effective . Neither mutant Alk nor mutant Alk were capable to rescue SB induced defects at doses of as much as pg mRNA mTOR signaling pathway . Mesodermal marker gene expression was also rescued by Alk SM. Whereas SB taken care of Alk WT embryos showed little or no gsc at shield stage, expression was restored in Alk SM embryos . Similarly, ntl expression from the dorsal margin was existing in SB treated Alk SM embryos but not in Alk WT embryos . The notochord expression domain of ntl was restored in SB handled Alk SM embryos, but not in Alk WTembryos Midline expression of shh and axial at bud stage in taken care of embryos could also be rescued by injection of Alk SM . With the somite stage, the full array of expression of shh, including the anteriormost domain, was restored by Alk SM but not by Alk WT .
Alk SM rescues phenotypic and gene expression selleckchem inhibitor defects in SB taken care of Xenopus embryos In Xenopus embryos treated with SB in advance of gastrulation, we located that expression of Alk SM rescues blastopore lip formation . We also observed, even so, that doses of Alk mRNA necessary for rescue of blastopore lip formation in Xenopus induce selleck Tideglusib extra, inhibitorindependent defects in post gastrula patterning. This observation suggests that ectopic Alk expression might possibly be sufficient to alter nodal signaling patterns in Xenopus embryos. A earlier report also noted weak mesodermal induction by wild type Alk injection alone , steady with this particular chance. We think that this may perhaps be since embryos are exquisitely delicate to levels of nodal signaling during gastrulation, and that more Alk may result in excessive signaling by endogenous ligands.

Endothelial cells have been isolated from human middle cerebral a

Endothelial cells were isolated from human middle cerebral arteries as previously described . Briefly, segments of human middle cerebral arteries collected following quick autopsies were embedded in Al of Matrigel in effectively plates and covered with endothelial basal medium supplemented with fetal bovine serum and penicillin streptomycin fungizone mixture. Following days in culture using the medium becoming changed each and every days, Matrigel containing microvessel outgrowths from human middle cerebral arteries had been dissected together with the help of an inverted microscope and dissociated a few instances in EBM by a sterile pipette tip. Matrigel fragments were then plated on plastic culture flasks, and incubated in EBM at C, CO with medium changed each days. Immediately after to days in culture, cells have been subjected to a double immunostaining with an antibody against aspect VIII and an antibody against a smooth muscle actin so as to confirm their endothelial nature Measurement of human brain endothelial cells viability and proliferation Main cultures of human brain endothelial cells have been plated at a density of cells AL of EBM containing of fetal bovine serum in wells culture plates and treated with many different doses of h and g secretase inhibitors as indicated from the figure legends.
Following h in culture, the EBM covering the cells was removed and assayed for Lacticodehydrogenase action working with the cytotoxicity detection kit . Cells had been selleck chemical Entinostat price covered with AL of EBM supplemented with fetal calf serum and cellular proliferation measured applying the Rapid cell proliferation assay kit Capillary morphogenesis assay Two hundred microliters of Matrigel was placed into every very well of a well culture plate at C and allowed to polymerize by incubation at C. Human middle cerebral artery endothelial cells had been seeded to the Matrigel in ml of EBM containing fetal calf serum. The cells had been incubated at C for h in the humidified CO atmosphere in the presence or absence of diverse doses of h and g secretase inhibitors as indicated inside the figure legends CH CH and P P statV which can be not cell permeable have been handled using the Projecti protein transfection reagent kit and management disorders had been established working with exactly the same dose of Pro jecti protein transfection reagent alone applying the manufacturer?s recommendations .
The experiments had been performed in quadruplicate for every therapy problem. For every culture, randomly selected fields had been photographed using a aim. An experimenter unaware of your various therapies measured the total length of tube structures in just about every photograph implementing the Image Pro Plus software package . Capillary network lengths for the several treatment chlorpheniramine problems had been expressed since the percentage of capillary network lengths obtained in the handle condition. Twenty four nicely tissue culture grade plates were covered with AL of Matrigel and permitted to gel for min at C, CO.

ATM is a Ser Thr protein kinase that’s activated in response to D

ATM is really a Ser Thr protein kinase that is activated in response to DNA doublestrand breaks and can phosphorylate a number of substrates involved in cell cycle checkpoint handle and DNA repair . ATM is held inactive in non irradiated cells as being a dimer or even a greater buy multimer. Cellular irradiation induces quick intermolecular autophosphorylation of Ser, which leads to dimer dissociation and initiates cellular ATM kinase action . Activated ATM can phosphorylate p at Ser, which increases its stabilization and nuclear accumulation and also its transactivation . It’s been reported that the IRinduced cell cycle stage specificity of ATM activation and p Ser phosphorylation is obvious. This promptly enhances their action in regular human lymphoblastoid cells, but just isn’t accompanied by a adjust during the abundance with the ATM protein . Our observations regularly indicate that the administration of emodin induces a rapid ATM phosphorylation at Ser and subsequently results in p Ser phosphorylation.
Additionally, however the ATM siRNA cannot totally knockdown the expression of ATM, we nevertheless found the decrease of ATM expression could attenuate emodininduced p accumulation as well as the level of phospho p. Furthermore, both ATM and p phosphorylation are blocked from the radical scavenger ascorbic acid. These findings support the notion that ATMdependent p activation is involved with emodin selleck chemicals VX-680 clinical trial elicited apoptosis. Survivin, a member with the inhibitor of apoptosis protein family members , has been characterized as being a p repressed gene. Additionally, p has been shown to bind to a consensus p binding web-site around the survivin promoter in vivo, which raises the possibility that p represses survivin at the transcriptional level . A preceding examine showed that reduction of wild sort p perform in tumor cells may possibly contribute to your upregulation of survivin and selleckchem inhibitor resistance to DNA damaging agents . Within the existing research, we discovered that the emodin mediated apoptosis is accompanied from the down regulation of survivin and activation of p, of which the knockdown of p recovered the expression of survivin in emodin handled cells.
The amounts of other IAP loved ones molecules which include cIAP and XIAP, yet, weren’t impacted by emodin . These observations indicate that a reduce of survivin could sensitize cells to emodin mediated cytotoxicity through a p read full article dependent pathway. In conclusion, this is actually the primary research to demonstrate that emodinmediated reactive oxygen species manufacturing stimulates ATM phosphorylation and activation, which then induces the phosphorylation of p. These two phosphorylation occasions perform critical roles in emodin induced apoptosis. Based on these observations, it’s obvious that emodin almost certainly exerts its cancer preventive therapeutic results immediately by means of the reactive oxygen species ATM p Bax signaling pathway, using mitochondria as being a universal important effector of cell death.

Pre treatment with wortmannin absolutely abolished the protective

Pre treatment method with wortmannin fully abolished the protective results of NDMC on HO induced apoptosis Discussion The outcomes reported within the existing study display that NDMC activates Akt and promotes the consequent inhibitory phosphorylation of GSK with the activation of opioid receptors. The involvement of opioid receptors in NDMC regulation of Akt and GSK was documented not only in CHO DOR cells, but also in NG cells and while in the rat nucleus accumbens, indicating that it had been not dependent on receptor overexpression or a individual cellular context. Additionally, in NG cells NDMC was discovered to exert a wortmannin delicate protective impact against oxidative strain, suggesting that theNDMC regulation of PIK Akt GSK signalingmay have important consequences for that management of cell survival. In CHO DOR cells, experiments aimed to investigate the sequence of events top rated to Akt and GSK phosphorylation indicated the involvement of PTX sensitive Gi Go proteins and the requirement of Src and IGF receptor tyrosine kinase actions, as demonstrated through the blockade induced by PP, but not PP, and tyrphostin AG .
Activation of a number of GPCR is shown to boost the action of selleck chemicals MK 801 Src relatives tyrosine kinases and Src continues to be demonstrated for being a vital regulator of GPCR action, modulating receptor internalization, desensitization and coupling to ERK and RTK . Evidence has also been presented the G protein subunits G i and G s, but not G q, G or G ?, can immediately activate Src . Prior research have demonstrated that Src can regulate IGF I receptor and that Src kinase can substitute to the receptor kinase in phosphorylating and activating IGF I receptor . We uncovered that CHO DOR cell remedy with NDMC enhanced the tyrosine phosphorylation of immunoprecipitated IGF I receptor in a PP dependent manner. In addition, NDMC elevated IGF I receptor phosphorylation at Tyr and Tyr, two tyrosine residues situated during the receptor kinase domain.
Phosphorylation of these residues is recognized to get necessary for ligand induced receptor kinase activation, and can also be induced by Src . Hence, a attainable explanation on the present findings is NDMC, by stimulating Gi coupled opioid receptors, promoted the Src dependent transactivation of IGF I receptor, with the consequent recruitment and activation of PIK. In help of this likelihood, inhibition of PIK activity by either Sympatol wortmannin or LY enormously suppressed NDMC induced Akt and GSK phosphorylation, indicating that PIK plays a essential function within this response. Mammalian cells have numerous PIK isoforms with diverse substrate specificity and distinct regulatory mechanisms .

We also find that scratching leads to the improved quantities of

We also find that scratching leads to the enhanced quantities of cyclin D, that is promoted by catenin more than expression and accountable for cell proliferation. These findings supply a achievable mechanism implicated while in the injury and fix of airway epithelium. Materials and methods Materials Protease XIV, insulin, transferrin, hydrocortisone, epidermal growth issue , retinoic acid and bovine serum albumin have been from Sigma Aldrich . Protease Inhibitor Cocktail was obtained from Calbiochem . FITC conjugated goat anti mouse secondary antibody, NE PER? Nuclear and Cytoplasmic Extraction Regents, BCA kit and Enhanced chemiluminescence have been bought from Pierce Chemical Enterprise . The antibodies, together with GSK , PKC?, catenin, tubulin, lamin B, cyclin D, mouse IgG, horseradish peroxidase conjugated secondary antibody and protein A agarose beads were the products of Santa Cruz Biotechnology, Inc The phosphorylated GSK , PKB antibody and the PI K inhibitor LY have been the items of Cell Signaling Technological innovation Inc The PKC inhibitor GFX was obtained from BioSource Worldwide Inc Lipofectamine was purchased from Invitrogen Daily life Technology .
Luciferase assay kit and galactosidase assay kit had been the solutions of Promega Corporation . Nocodazole were purchased from Sigma Aldrich . Plasmids and transient transfection The constitutively activated GSK mutant was generously provided by Professor J.R. Wooggett . The secure mutant catenin pCSMMBCSAMT was generously provided by Dr. Tofacitinib Rolf Kemler . Tcf luciferase reporter plasmids had been generous presents from Dr. Bert Vogelstein . Each construct harbors an Xho fragment containing three copies of wild variety or mutant human Tcf binding website cloned into pGL Standard plasmid. Transient transfection of the plasmids described over was carried out by using Lipofectamine in accordance with the recommendation from producer and a method described by Tucker et al. with minor modification. Cell culture Porcine bronchial epithelial cells were prepared as previously described .
Briefly, the bronchi was resected from freshly slaughtered pigs, rinsed with selleckchem inhibitor cold D Hanks answer containing antibiotics , and filled with . protease XIV resolution followed by incubation at C for about h with PHA-848125 gentle shaking. The protease remedy was collected, and bronchi had been intensively washed with DMEM F containing antibiotics and new calf serum. The washing choice was centrifuged together with the protease remedy to gather cells. The cells had been washed once alot more with all the washing resolution described above prior to resuspension in comprehensive culture medium, which was DMEM F supplemented with g ml insulin, g ml transferrin g ml hydrocortisone, ng ml epidermal development issue , mmol L retinoic acid mg ml BSA, fetal bovine serum , and antibiotics .

The totally free radical NO can trigger the induction of two inde

The free radical NO can set off the induction of two independent apoptotic pathways that consist of nuclear DNA degradation plus the publicity of membrane phosphatidylserine residues . Degradation of DNA at once impacts cellular survival , but the exposure of membrane PS residues can play a much more formidable position by leading to cellular irritation, thrombosis , and microglial phagocytosis of viable cells . Closely linked to cellular NO toxicity could be the induction of mitochondrial membrane depolarization and also the activation of particular caspases which might be regarded as to become important for genomic DNA degradation and membrane PS externalization. Ahead of mitochondrial membrane depolarization and the subsequent release of cytochrome c, caspase precipitates the activation of caspase also as caspase through the intermediary caspase . Collectively, caspase and caspase result in the two DNA fragmentation and membrane PS publicity .
This cascade of events could very well be tempered by the increased expression of the Bcl household member Bcl xL to prevent cytochrome c release and cellular apoptosis Offered the possible central part that Akt may well hold while in vascular injury, we examined a lot of the important regulatory factors that have been both needed and ample for Akt to modulate genomic DNA integrity, membrane PS publicity, and microglial activation. Products and strategies Cerebral microvascular endothelial cell selleck TG 100713 cultures Vascular ECs were isolated from Sprague Dawley grownup rat brain cerebra through the use of a modified collagenase dispasebased digestion protocol . Briefly, ECs had been cultured in endothelial development media consisting of ME with heat inactivated fetal bovine serum, mM L glutamine, Ag ml heparin, and Ag ml EC development supplement . Experiments were carried out with cells in the third passage. Cells had been recognized as endothelial by a cobblestone physical appearance with phase contrast microscopy, have been positive with direct immunocytochemistry for aspect VIII connected antigen, and were unfavorable for GFAP immunocytochemistry. Following three passages, cells had been purity for ECs.
Steady transfection of myr Akt cDNA or dominant adverse Akt cDNA construct into ECs Steady EC clones overexpressing the myristoylated Pimobendan form of Akt were generated by transfecting the cells which has a cDNA construct beneath the handle of the CMV promoter with cDNA containing sequences corresponding to amino acids of avian c rsc in the V finish in addition to a Myc His tag with the Vend on the mouse Akt open reading through frame by lipofection with Lipofectamine Plus reagent .

For quantification of apoptosis, a complete of no less than , aci

For quantification of apoptosis, a total of not less than , acinar cells were counted on pancreatic tissue sections or cell smears for every situation. Statistical analysis of data This was accomplished by utilizing two tailed Student’s t check. p value . was thought to be statistically vital. Effects Changes in pancreatic levels of Bcl proteins in designs of acute pancreatitis Western blot examination showed the prosurvival proteins BclxL and Bcl have been present in normal rat and mouse pancreas, and had been up regulated in rodent versions of acute pancreatitis . Up regulation of pancreatic Bcl xL protein was detected in all models examined, namely pancreatitis induced by cerulein in rats and mice, by L arginine in rats, and by cholinedeficient ethionine supplemented food plan in mice. The extent of Bcl xL up regulation in fully formulated pancreatitis was maximal from the rat cerulein model , and minimal while in the rat L arginine model . In a different way, pancreatic Bcl degree enhanced markedly in rat cerulein pancreatitis but not other models .
The upregulation of Bcl xL and Bcl occurred early within the improvement of cerulein pancreatitis, remaining already evident min after the induction of pancreatitis . Pancreatic ranges on the critical pro apoptotic protein Bax didn’t change in the versions of pancreatitis tested . One more major pro apoptotic Bcl protein, Bak, was markedly upregulated inside the rat L arginine model, and also to a smaller sized extent, in mouse and rat cerulein pancreatitis . We also measured the ranges of professional apoptotic selleck informative post BH only proteins, Bim and Bid, in designs of pancreatitis induced by cerulein in rat and mice. Rat cerulein pancreatitis is characterized by greater apoptosis and lower necrosis, whereas mouse cerulein model has low apoptosis and high necrosis . Western blot evaluation showed no improve in Bim ranges in these models of pancreatitis , indicating against its leading function while in the regulation selleckchem inhibitor of cell death in pancreatitis. The ranges of Bid have been also low to detect each in normal pancreas and in versions of pancreatitis.
Bcl xL and Bcl amounts in pancreatic mitochondria increase in the course of cerulein pancreatitis Death responses are regulated by Bcl proteins localized from the mitochondria . Therefore, a significant question read what he said is no matter if the increases in pancreatic ranges of Bcl xL and Bcl that we observed in versions of pancreatitis translated into corresponding increases in mitochondrial levels of those proteins. For these measurements we used pancreatic mitochondria isolated from rats and mice as we have lately described in detail . We also showed that as compared to total tissue homogenates, mitochondrial preparations have been enriched in mitochondrial marker cytochrome c oxidase IV, contained significantly less ER marker calnexin, and no cytosolic marker LDH .

This does not preclude the likelihood that this might possibly ha

This isn’t going to preclude the probability that this could possibly have occurred during the original isolation with the clones . Interestingly, more cells in the clone cell line were able to enter mitosis a second time when compared with the parental HCT cells. The basis of this difference is now identified. Given that the presence of p slows down re replication and appeared to decreased the quantity of colonies immediately after ZM therapy,we analyzed p responses in a number of the cell lines that arose after therapy of HCT p cells with ZM. All but a single cell line showed a usual induction of p protein in response to Etoposide and ZM . The defect in Clone will not appear to be as a result of alteration from the hDM mediated degradation of p given that the hDM inhibitor Nutlinwas ready to induce p . Also, p in Clone was even now phosphorylated at serine in response to Etoposide indicating that DNA damage signaling pathways upstream of p may well be intact . For this reason, the emergence of colonies is simply not necessarily associated using the alteration of p signaling pathways.
Asymmetric division in ZM treated cells The presence of cells capable of proliferating following the elimination of Aurora kinase inhibitors is probably related to the clinical response to this class of agents. Human tumor cells attempt mitosis many times within the presence of ZM and obtain massive amounts of DNA , at some point starting to be giant and multinucleated. selleck supplier IOX2 One particular way that clones may well emerge right after ZM remedy is for your giant cells to undergo asymmetric cell division, therefore creating smaller sized viable cells. To begin to tackle this idea we determined whether or not human tumor cells had been capable of proliferating following getting rid of ZM. HelaM cells were exposed to MZM extended adequate to permit just one failed attempt at mitosis. The drug was removed and cell fate was established by time lapse microscopy. Cells handled on this manner were capable to enter mitosis and divide as lots of as four occasions in advance of the finish of your experiment . Below these circumstances, attempts at mitosis usually created three cells, or two cells of different sizes.
This indicates that ZM is reversible in vivo. Subsequent, we made use of time lapse microscopy to monitor giant HCT cells created by longer therapy with ZM and after that replated inside the absence from the drug. A lot of the multinucleated giant cells died during the filming practice, steady using the Lacosamide reduced charge of colony formation. Some giant cells had been able to enter mitosis and, on mitotic exit, formed various cleavage furrows . The presence of condensed chromosomes confirms that these had been in reality mitotic events . In some cases cleavage was thriving and asymmetrical . To measure the frequency of asymmetric division, HCT? ? cells have been exposed to ZM until they’d progressed as a result of mitosis 3 times .