Number Of Bcl-2 Positive Cells Research Articles

BCL-2 Is Expressed in Advanced Mastocytosis and Midaustorin Induces Venetoclax Sensitivity of Mast Leukemia Cell Lines

*° contributed equally to this work. Introduction: Systemic Mastocytosis (SM) is a heterogeneous disorder characterized by mast cells (MCs) accumulation in various tissues and associated with KIT mutations (KIT D816V) in more than 90% of the cases. It includes indolent (ISM) and advanced diseases (advSM), which are associated with additional molecular abnormalities. For advSM, recent clinical studies have shown that Midostaurin, a kinase inhibitor of WT and mutant KIT, induces high rate of responses associated with significant improvement of prognosis. However, complete responses are infrequent and relapses occur in a significant proportion of patients. Therefore, combination therapies are needed to increase overall response rate and prevent relapses. Venetoclax is a selective orally bioavailable BCL-2 inhibitor that induces cell death and is currently used for treatment of various lymphoid and myeloid malignancies. In an attempt to identify novel diagnostic and prognostic markers and potentially new therapeutic targets for mastocytosis, bone marrow sections of patients with different categories of mastocytosis were analyzed by IHC using anti-BCL-2 antibodies. BH-3 profiling was used to assess BH-3 proteins dependency, and sensitivity to Venetoclax alone or in combination of Midostaurin. Methods: Thirty-three adult patients were included in this preliminary study. According to the WHO classification, patients were classified as having ISM (n=10), Smoldering SM (SSM n=1), advSM (n=16, including SM-AHN (n=9), MC leukemia (MCL n=4), MC sarcoma (MCS n=2)). Most patients were KIT D816V (n=30; 90.9%); two MCL and one MCS exhibited extracellular and juxtamembrane mutations, respectively. Among these patients, 9 were treated with Midostaurin as first line therapy. Formalin fixed bone marrow sections were performed at diagnosis and during follow up. Mast cells were identified by Giemsa staining and as CD117 and tryptase positive cells. BCL-2 staining was performed by immunohistochemistry in formalin paraffin embedded fixed section. BCL-2 staining was considered as positive (>5%), heterogeneous (partial staining) or homogeneous (>80% positive cells), of high or low intensity (> or = or < to residual T cells). BH3 profiling was performed in ROSA KIT WT and ROSA KIT D816V using Cytochrome C upon exposure to distinct BH3 peptides/mimetics. Results: In ISM, BCL-2 staining was negative (n= 2/10) or when positive only in rare MCs (n=8/10), with low intensity. In contrast, all advSM cases were positive (16/16) with high (13/16), and homogeneous (6/16) staining. In MCL and MCS, BCL-2 staining was always positive with a homogeneous and high staining. In patients treated with Midostaurin, BCL-2 staining was performed before and three months after treatment initiation. Although MCs infiltration was reduced at least by 50% in all cases, number of BCL-2 positive cells and intensity of staining remain unchanged. In vitro, flow cytometry analysis showed that both MCL-like cell lines (ROSA KIT WT and ROSA KIT D816V) expressed BCL-2, MCL-1 and BCL-XL proteins. When treated with Midostaurin (200nM) for 48 hours, expression of BCL-XL and MCL-1 significantly decreased in MC lines especially the one with KIT D816V mutation. Interestingly, BCL-2 expression remained unchanged upon Midostaurin treatment, which was consistent with in vivo observations. Dynamic profiling performed in ROSA cell lines revealed that priming by midostaurin dramatically enhanced apoptotic dependencies to BCL-2 and other BH-3 proteins (>20% of apoptosis), especially in ROSA KIT D816V (figure). Conclusion: High expression of BCL-2 is associated with advSM and may participate to the pathogenesis of the disease, to its resistance to conventional chemotherapies and to partial resistance to Midostaurin. Consistent with its effect in reducing MCL-1 and BCL-XL expression, Midostaurin restored apoptotic dependency to BCL2 in human MCL-like cells, thereby suggesting that midostaurin could sensitize mast cell tumor to venetoclax. Our results provide thus a rationale to use a combination of Midostaurin and Venetoclax to treat AdvSM patients. Figure Disclosures Dubreuil: AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. OffLabel Disclosure: Venetoclax preclinical studioes on mastocytosis

Electroacupuncture Ameliorates Cerebral Ischemia-Reperfusion Injury by Regulation of Autophagy and Apoptosis.

Background. The therapeutic mechanisms of cerebral ischemia treatment by acupuncture are yet not well addressed. Objective. We investigated the effects of electroacupuncture (EA) at GV26 observing the expression of autophagy-related proteins Beclin-1 and LC3B and proportion of apoptotic cells and Bcl-2 positive cells in MCAO/R model rats. Methods. Sprague-Dawley (SD) male rats were randomly assigned to 7 groups: model groups (M6h, M24h, and M72h), EA treatment groups (T6h, T24h, and T72h), and sham operation group (S). Neurological deficit and cerebral infarction volume were measured to assess the improvement effect, while the expression of Beclin-1 and LC3B and proportion of Tunel-positive and Bcl-2 positive cells were examined to explore EA effect on autophagy and apoptosis. Results. EA significantly decreased neurological deficit scores and the volume of cerebral infarction. Beclin-1 was significantly decreased in T24h, while LC3B-II/LC3B-I ratio markedly reduced in 6th hour. EA groups markedly reduced the number of Tunel positive cells, especially in T24h. Meanwhile, the number of Bcl-2 positive cells obviously increased after EA treatment, especially in T6h and T24h. Conclusions. The alleviation of inadequate autophagy and apoptosis may be a key mechanism involved in the reflex regulation of EA at GV26 to treat cerebral ischemia.

단삼(丹蔘)이 뇌조직출혈 흰쥐의 신경세포 자연사에 미치는 영향

Objects : The purpose of this study was to observe the effect of Salviae Miltriorrhiza Radix(SMR) water-extract on intracerebral hemorrhage(ICH) and neuronal apoptosis in the injured areas. Method : ICH was induced by the stereotaxic intrastriatal injection of bacterial collagenase type IV in Sprague-Dawley rats. The rats were givened oral SMR treatment once a day for three days after the ICH treatment. TUNEL positive cells in the affected regions were performed by TUNEL assay, Bax and Bcl-2 positive cells by immunohistochemistry and the Bax expression by western blotting method. Results : The results are as follow; 1. SMR significantly reduced the number of TUNEL positive cells in the peri-hematoma reigions of ICH-induced rats. 2. SMR significantly reduced the number of Bax positive cells in the peri-hematoma regions of ICH-induced rats. 3. SMR did not affect the number of Bcl-2 positive cells in the peri-hematoma regions of ICH-induced rats. 4. SMR significantly reduced the Bax expressions compared with ICH group in hemorrhagic hemisphere of ICH-induced rats. Conclusion : These results suggest that SMR is effective in reducing neuronal apoptosis.

Differential expression of Bcl-2 and Bax during gastric ischemia-reperfusion of rats

To investigate expression of Bcl-2 and Bax in gastric ischemia-reperfusion (GI-R) and involvement of extracellular signal-regulated kinase (ERK) 1/2 activation. The GI-R model was established by ligature of the celiac artery for 30 min and reperfusion in Sprague-Dawley rats. Rats were assigned to groups in accordance with their evaluation period: control, 0, 0.5, 1, 3, 6, 24, 48, and 72 h. Expression and distribution of Bcl-2 and Bax proteins were analyzed by immunohistochemistry and western blotting in gastric tissue samples after sacrifice. Compared with controls, the percentage of positive cells and protein levels of Bcl-2 decreased in the early phases of reperfusion, reached its minimum at 1 h (P < 0.05); it then increased, reaching its peak at 24 h of reperfusion (P < 0.05). The pattern of Bax expression was opposite to that of Bcl-2. Bax expression increased after reperfusion, with its peak at 1 h of reperfusion (P < 0.05), and then it decreased gradually to a minimum at 24 h after reperfusion (P < 0.05). On the other hand, inhibition of activation of ERK1/2 induced by PD98059, a specific upstream MEK inhibitor, had significant effects on Bcl-2 and Bax in GI-R. Compared with GI-R treatment only at 3 h of reperfusion, PD98059 reduced the number of Bcl-2 positive cells (0.58% of R3h group, P < 0.05) and Bcl-2 protein level (74% of R3h group, P < 0.05) but increased the number of Bax-positive cells (1.33-fold vs R3h group, P < 0.05) and Bax protein level (1.35-fold of R3h group, P < 0.05). These results indicated that the Bcl-2 and Bax played a pivotal role in the gastric mucosal I-R injury and repair by activation of ERK1/2.

Increased butyrate formation in the pig colon by feeding raw potato starch leads to a reduction of colonocyte apoptosis and a shift to the stem cell compartment

Whereas butyrate is well known to induce apoptosis in transformed colon cells in vitro, evidence exists that it inhibits apoptosis of colon crypt cells in vivo. In this study, pigs were fed with resistant potato starch to increase microbial butyrate formation in the colon and to investigate its effects on mitosis and apoptosis. In addition, apoptosis regulating proteins were determined by immunocytochemistry, such as proapoptotic Bak, antiapoptotic Bcl-2, and the epidermal growth factor (EGF), which is synthesized by goblet cells and functions as a survival factor. Two groups of 6 barrows were both supplied with 381 g crude protein and 31 MJ metabolizable energy (ME) daily over a 19-day experimental period. The rations differed in the carbohydrate composition. The controls received gelatinized starch as the main carbohydrate, whereas the experimental group (butyrate group) received a ration with raw potato starch (low ileal digestibility). In the feces, butyrate concentration and pH were monitored daily. After killing the pigs, colon tissue was obtained for histologic and immunocytochemical evaluation, which was performed separately in the luminal, middle, and stem cell compartment of the crypts. In the butyrate group, the total number of apoptotic cells was reduced by 34% (P ≤ .001) compared with controls, whereas the mitotic rate was not altered. The crypt depth was only moderately increased by 15%. Apoptosis in the luminal compartment of the butyrate group was reduced by 18.8%, but was increased by 21.7% in the stem cell compartment. The effect of butyrate on apoptosis was paralleled by an increased number of Bcl-2 positive cells mainly in the luminal compartment (butyrate: 2.6 cells; controls: 1.2 cells, P ≤ .001), which was more pronounced compared with the number of Bak positive cells in the same compartment. Bak activity in the stem cell compartment was 3.4-fold increased compared with controls (P ≤ .001). The size of EGF-positive stained mucus-droplets from the goblet cells was increased in the butyrate group (P ≤ .001). We conclude that butyrate inhibits apoptosis of colonocytes in vivo. An excessive proliferation of crypts is counteracted by a shift of the remaining apoptosis towards the stem cell compartment.

Expression of apoptosis-related proteins and morphological changes in a rat tumor model of human small cell lung cancer prior to and after treatment with radiotherapy, carboplatin, or combined treatment.

In order to understand the apoptosis pathway in tumor cells, differences in cell morphology and expression of apoptosis-related proteins induced by radiation and/or chemotherapy, were investigated in a rat double tumor model comparing cisplatin-sensitive and -resistant human small cell lung cancer tumors. The cisplatin-sensitive human small cell lung cancer cell line (GLC4) and its cisplatin-resistant subline (GLC4-CDDP) were each injected subcutaneously in a different hind limb of the rat. After 15-17 days, radiation (10 Gy), carboplatin (25 mg/kg, intraperitoneal), combined treatment, or no treatment was administered. In combined treatment, carboplatin was administered 24 h before radiation. Tumors were removed and fixed 72 h after carboplatin or 48 h after radiation. Paraffin-embedded slides were stained with hematoxylin/eosin for morphology. Expression of the apoptosis-related proteins p53, p21, Rb, bcl-2, bax, c-myc, Fas and Fas-L was assessed immunohistochemically. In the GLC4 tumor, carboplatin treatment increased tumor cell size, tumor cell size heterogeneity, and number of apoptotic tumor cells. After radiation and combined treatment, these changes were more pronounced and multinuclear giant cells were observed. In GLC4-CDDP tumors, minimal changes were detected after carboplatin. Following radiation slight increases in tumor cell size, tumor cell size heterogeneity and number of apoptotic tumor cells were observed. No multinuclear giant cells were seen. After combined treatment, GLC4-CDDP showed cellular changes comparable to those in GLC4 cells after radiation or combined treatment, but no giant cells were observed. Untreated, both tumors were equally positive for p53, bax, Fas, Fas-L, c-myc and negative for Rb. Contrary to GLC4, untreated GLC4-CDDP tumors showed no p21 and bcl-2 expression. After combined treatment, an increase in number of bcl-2 positive cells was found in GLC4-CDDP tumors. No p21 was induced in GLC4-CDDP by any treatment modality. In GLC4 tumors, p21 was induced by radiation alone. No further changes in protein expression were induced by any treatment in GLC4 tumors. Following therapy, morphological changes in cell size and cell size heterogeneity were more pronounced in GLC4 than in GLC4-CDDP tumors. However, morphological changes in GLC4-CCDP tumors after treatment indicate that carboplatin plus radiotherapy may be effective also in cisplatin resistant tumor cells. An increase in p21 in GLC4 after radiation might facilitate apoptosis. The increase in number of Bcl-2 positive cells after combined treatment and the consistently negative p21 status after any treatment in GLC4-CDDP may protect these tumor cells from apoptosis as a part of their resistance mechanism to cisplatin.

Progressing imbalance between proliferation and apoptosis with increasing severity of cervical intraepithelial neoplasia.

The equilibrium between cell proliferation and protection against apoptosis was studied immunohistochemically using monoclonal antibodies against Ki-67-Ag and bcl-2, respectively, in consecutive sections from normal and metaplastic cervical epithelia and cervical intraepithelial neoplasia (CIN) lesions and cervical carcinomas. A high percentage of Ki-67-Ag positive cells was seen in the parabasal cells of normal ectocervical and mature squamous metaplastic epithelium, although the basal cells were virtually negative. In preneoplastic lesions, however, the basal cells showed high proliferative activity and an increasing frequency of Ki-67-Ag positive cells was observed in the higher epithelial layers with increasing severity of CIN. In squamous cell carcinomas, variable numbers of Ki-67-Ag positive cells were observed and in adenocarcinomas expression increased with the degree of anaplasia. bcl-2 expression was observed only in the basal cells of normal endo- and ectocervix including reserve cells. With increasing severity of CIN, staining intensity and number of bcl-2 positive cells gradually increased. Five of eight squamous cell carcinomas were variably positive. All five adenocarcinomas showed extensive bcl-2 expression. Increased expression of both Ki-67-Ag and bcl-2 with increasing severity of CIN indicates an increasing imbalance between cell proliferation and protection from apoptosis. It is therefore proposed that an increasing proliferative fraction combined with a higher number of cells protected from apoptotic cell death contributes to progression of CIN. This phenotype may identify premalignant lesions with the potential to transform to cervical cancer.