Increased prostate-specific membrane antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against bcl-2 and EGFR

Antisense oligonucleotides have been employed against in vivo and in vitro prostate cancer models. While most oligos consist of a single mRNA binding site, targeting a single gene product or others sharing sequence homology, our laboratory has developed bispecific oligos directed toward even unrelated proteins. This study evaluates the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 [the second bispecific binding site was directed against the epidermal growth factor receptor (EGFR)]. Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) were subsequently evaluated. When LNCaP prostate tumor cells were incubated with bispecific oligos (directed against bcl-2 and EGFR) and compared to lipofectin-containing controls significant growth inhibition resulted. In subsequent experiments, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with the monospecific directed solely against bcl-2. No differences were detected in mRNA levels encoding PSA following treatment with either mono- or bispecific oligos. Previously, we suggested that cell growth inhibition produced by some bispecifics could be attributed to complementary double-stranded regions formed by intra-strand base pairs. Double-stranded nucleic acids are known inducers of interferon, which promote expression of cell surface HLA type antigens. If induced, perhaps this cytokine also enhances PSMA expression, making prostate tumor cells a more recognizable target for cytotoxic T cells.     

Differentiated prostatic antigen expression in LNCaP cells following treatment with bispecific antisense oligonucleotides directed against BCL-2 and EGFR

Antisense oligonucleotides (oligos) have been administered against in vivo and in vitro prostate cancer models employing LNCaP and PC-3 cell lines. While most oligos consist of a single mRNA binding site targeting a single gene product or those with sequence homology, our lab has developed bispecific oligos directed toward two unrelated proteins. In LNCaP cells, we initially identified bispecifics that increased the expression of prostate-specific membrane antigen (PSMA) while not affecting secreted prostate-specific antigen (PSA). We postulated that surface antigen expression is increased by bispecifics able to form double-stranded regions, acting as interferon (IFN-γ) inducers. In other systems, when induced, IFN-γ promotes cell surface antigen expression, including HLA and receptors for tumor necrosis factor. To test this hypothesis, we measured the effect of oligo treatment on both IFN-γ induction and the expression of another secreted product of differentiated prostate cells, prostatic acid phosphatase (PAP). This study initially evaluated the inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against bcl-2 (the second bispecific binding site was against the epidermal growth factor receptor). Employing RT-PCR, the expression of non-targeted proteins encoded by mRNA for PSMA, PSA, PAP, and IFN-γ was subsequently valuated. When LNCaP prostate tumor cells were incubated with oligos and compared to lipofectin-containing controls significant growth inhibition resulted. Employing RT-PCR, the levels of mRNA encoding PSMA were unexpectedly found to be elevated following treatment with the bispecific oligos but not with a monospecific directed solely against bcl-2. No differences were detected in mRNA levels encoding PSA following treatment with either oligo type. IFN-γ was significantly induced only by bispecific oligos, and PAP expression was similar to PSA. These data support the hypothesis that double strand-forming bispecific oligos induce IFN-γ that enhances cell surface PSMA expression. Expression of tumor-associated surface antigens could increase their recognition and targeting by immunologic defense mechanisms and increase the effectiveness of tumor vaccines.     

Multigene targeting of signal transduction pathways for the treatment of breast and prostate tumors

Previous studies have demonstrated that monospecific antisense oligonucleotides (oligos) directed against mRNA encoding proteins associated with tumor growth, death, and survival are efficacious against breast and prostate tumors. Targeted proteins, associated with different signal transduction pathways, have included transforming growth factor-alpha [TGF-α (MR₁)], its binding site the epidermal growth factor receptor [EGFR (MR₂)] sharing sequence homology to the breast cancer prognostic marker Her-2/neu, an apoptosis inhibiting protein [bcl-2 (MR₄)], and the androgen receptor [AR (MR₅)]. In attempts to enhance antisense therapy, recent reports describe how two of the binding sites mentioned above can be sequentially placed within a single complementary (bispecific) strand and administered either in the presence or absence of additional therapeutic agents. When tested against breast and prostate tumor cell lines specific differences were noted: MCF-7 breast cancer cells were more receptive to the inhibitory effects of monospecific oligos, whereas PC-3 and LNCaP prostate cells were particularly responsive to bispecifics. In an effort to identify agents which enhance the activity of oligos and which possess less toxicity than traditionally employed chemotherapeutics, Rapamycin, an immunosuppressive agent known to regulate tumor growth and signal transduction mediated by the mTOR receptor, is compared to paclitaxel in combination therapy employing monospecific or bispecific oligos. Bispecifics were constructed recognizing the binding sites for TGF-α and EGFR mRNA [TGF-α/EGFR (MR₁₂) and EGFR/TGF-α (MR₂₁)]; another pair recognized binding sites for EGFR and bcl-2 [EGFR/bcl-2 (MR₂₄) and bcl-2/EGFR (MR₄₂)]; while a third pair employed only against the LNCaP prostate cell line recognized bcl-2 and the androgen receptor [bcl-2/AR (MR4₄₅) and AR/bcl-2 (MR₅₄)]. Oligo pairs differ in their 5′–3′ linear binding site orientations, and were tested in vitro against MCF-7 breast and PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were done for 2 days with the agents followed by 2 days in their absence. Five experiments evaluated the effect of monospecific or bispecific antisense oligos in combination with an LD₅₀ dosage of either Rapamycin or paclitaxel and led to the conclusion that although these agents act via different mechanisms, they are comparable in effectiveness.     

Treatment of MCF-7 breast cancer cells employing mono- and bispecific antisense oligonucleotides having binding specificity toward proteins associated with autocrine regulated growth and BCL-2

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha) (MR1) and its binding site, the epidermal growth factor receptor (EGFR) (MR2), are efficacious against the UACC 897 breast, PC-3 and LNCaP prostate, and T98G glioblastoma tumor lines in both in vitro and in vivo studies. Oligos against the anti-apoptosis protein bcl-2 (MR4) are also efficient against PC-3 and LNCaP tumors in similar in vitro experiments. To enhance activity, and also to introduce a derivative type of multifunctional oligo into this field, "bispecifics" were constructed containing two truncated complementary DNA sequences (from either MR1 or MR2) designed to bind targeted mRNA about their respective AUG initiation codons, and/or a similar sequence adjacent to the AUG site of mRNA encoding bcl-2. Tandem pairs of bispecifics were constructed: The first had complementary sequences for TGF-alpha and EGFR mRNA, but differed in 5' to 3' tandem orientation (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21] sequences); a second pair had binding sites associated with EGFR and bcl-2, also differing in orientation (EGFR/bcl-2 [MR24] and bcl-2/EGFR [MR42]). In studies targeting PC-3 and LNCaP cells, bispecifics demonstrated significant in vitro activity, and the second pair was significantly better than the original monospecifics. These studies are now extended to the MCF-7 breast cancer model in order to determine whether these particular bispecifics have similar anti-breast cancer activity and if they are significantly better than monospecific oligos from which they were derived. We conclude that bispecific oligos significantly inhibit MCF-7 growth, however, in contrast to results obtained with PC-3 and LNCaP, the monospecific oligos directed against EGFR and bcl-2 have significantly greater activity than the bispecifics targeting a combination of TGF-alpha, EGFR, or bcl-2. These data suggest that the relative activities of oligos, whether mono- or bispecific, change with tumor type. Bispecific oligos which target different proteins, possibly those which regulate estrogen utilization, may be more effective against MCF-7 cells and warrant additional investigation, particularly if co-administered with traditional chemotherapeutics.     

Bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-α) (MR₁) and its binding site, the epidermal growth factor receptor (EGFR) (MR₂), are efficacious against PC-3 and LNCaP prostate tumors. To enhance activity and aid in simultaneous delivery, “bispecific” 39-mer oligos were constructed containing portions of both MR₁ and MR₂ sequences. The first pair contained truncated sequences recognizing TGF-α and EGFR mRNA binding sites, about their respective AUG initiation codons. These bispecifics differ in their 5’ to 3’ tandem orientation (TGF-α/EGFR [MR₁2] and EGFR/TGF-α [MR₂₁] sequences). A second pair was constructed having complementary sequences for EGFR and bcl-2 (EGFR/bcl-2 [MR₂₄] and bcl-2/EGFR [MR₄₂]). All bispecifics were tested in vitro against PC-3 and LNCaP prostate tumor cells, and compared to mono-specific oligos from which they were derived. The purpose of this study was: (1) to evaluate bispecific antitumor activity; (2) to identify dominant sequences; (3) to identify effects of binding site orientation; and (4) to determine whether bispecifics are more effective when targeting one versus different growth-dependent pathways. Comparisons were made between oligos tested against either PC-3 or LNCaP cells incubated for 2 d with the agents followed by 2 d in their absence. The first PC-3 cell experiment demonstrated that bispecific MR₁2 and MR₂₁ oligos are at least as effective as their mono-specific counterparts and that the MR₂₁ bispecific orientation is more effective than the MR₁ mono-specific by 64% (p = 0.014). It also suggested that the sequence directed against EGFR contributed most to bispecific activity, particularly in the MR₂₁ orientation. In a second PC-3 study a second bispecific pair of 37-mer oligos was constructed containing bases complementary to mRNA encoding EGFR and the apoptosis-associated protein bcl-2 (MR₄). MR₂₄ was constructed with the EGFR complementary site at the 5’ end (EGFR/bcl-2), and MR₄₂, containing the opposite orientation (bcl-2/EGFR). Each contained the dominant EGFR activity identified previously. MR₁, MR₂, MR₄, MR₁2, MR₂1, MR₂4, and MR₄₂ (1X and 2X in concentration) were cultured with cells and compared to controls. Each oligo significantly inhibited growth of PC-3 cells. MR₄₂ was most effective and significantly better than MR₁ (p = 0.0128), MR₂ (p = 0.021), MR₄ (p = 0.0002), and MR₁2 (p = 0.0032). 2X MR₂4 and 2X MR₄₂ were better than their 1X concentration counterparts, but the differences were not significant. In a similar experiment MR₁, MR₂, MR4, MR₁2, MR₂1, MR₂4, and MR42 were cultured with LNCaP cells and compared to lipofectin-containing controls. Each oligo significantly inhibited the growth of LNCaP cells. Again, MR₄₂ was most effective and significantly better than MR₂ (p = 0.021) and MR₄ (p = 0.038). MR₂4 was significantly better than MR₂ (p = 0.048). Bispecific oligos are a significant advance in antisense technology and could play a role in treating prostate cancer, particularly if combined with traditional chemotherapeutics.     

Compensatory and non-compensatory effects on protein expression following BCL-2 suppression by antisense oligonucleotides

Antisense oligonucleotides (oligos) have been employed against in vivo and in vitro prostate cancer models targeting growth regulatory proteins. In LNCaP cells, we evaluated both monospecific and bispecific oligos that targeted and comparably suppressed the expression of bcl-2, an apoptosis inhibitory protein. Cells compensated with both suppressed caspase-3 (an apoptosis promoter) activity, and an enhancement of both androgen receptor (AR) and p300 expression. This suggests that a progression to increased androgen sensitivity accompanies bcl-2 suppression, in this tumor line. To further evaluate mechanisms of adaptation, we now evaluate the effects upon the expression of insulin-like growth factor (IGF1) and another AR coactivator, IL-4, thought to increase prostate cancer growth. IGF1 expression was not significantly altered suggesting this pathway need not be regulated when bcl-2 directed gene therapy is employed. In contrast to increased AR and p300 expression that compensated for bcl-2 suppression, the AR coactivator IL-4 expression was not increased, suggesting no role in any increased androgen sensitivity.     

Combination chemotherapy employing bispecific antisense oligonucleotides having binding sites directed against an autocrine regulated growth pathway and bcl-2 for the treatment of prostate tumors

In previous studies we demonstrated that antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-α [MR₁]), its binding site the epidermal growth factor receptor (EGFR [MR₂]), and the anti-apoptosis protein bcl-2 (MR₄) are efficacious against prostate tumors. In recent reports we also describe how two of these mRNA directed binding sites can be synthesized sequentially within a single linear complementary strand and administered either in the presence or absence of additional therapeutic agents. In these continuing experiments “bispecific” oligo pairs were further evaluated in the presence or absence of Cytoxan, Taxol, or DES. One oligo pair recognized the binding sites for TGF-α and EGFR mRNA (TGF-α/EGFR [MR₁₂] and EGFR/TGF-α [MR₂₁]); another pair recognized binding sites for EGFR and bcl-2 (EGFR/bcl-2 [MR₂₄] and bcl-2/EGFR [MR₄₂]). Oligo pairs differ in their linear 5′ to 3′ binding site orientations, and were tested in vitro against PC-3 and LNCaP prostate tumor cell lines. Following cell attachment, incubations were for 2 days with the agents followed by 2 days in their absence. When tested against PC-3 cells and combined with LD₅₀ Cytoxan, MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 47.3, 45.7, 68.3, and 64.9%; with LD₅₀ Taxol MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 49.8, 45.8, 64.1, and 59.2%; and with LD₅₀ DES MR₂, MR₄, MR₂₄, MR₄₂ significantly inhibited 66.6, 67.6, 64.3, and 67.2% respectively. Each agent significantly increased the inhibition produced by either oligo alone. LNCaP cells were also incubated with mono- and bispecific oligos in either the presence or absence of chemotherapeutics. MR₂, MR₄, MR₂₄, MR₄₂ produced significant inhibitions of 57.4, 58.4, 69.4, and 68.6% with LD₅₀ Cytoxan; 70.4, 70.1, 73.6, and 74.0% with LD₅₀ Taxol; and 49.8, 50.1, 59.6, and 53.9%, respectively with LD₅₀ DES. A complete PC-3 experiment compared MR₁, MR₂, MR₄, MR₁₂, MR₂₁, MR₂₄ and MR₄₂, in the presence of LD₅₀ Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR₁ by 51.0, MR₂ by 55.0, MR₄ by 58.0; MR₁₂ by 56.0; MR₂₁ by 61.1, MR₂₄ by 65.5 and MR₄₂ by 66.0%. Bispecifics directed against two different pathways, MR_24, and MR₄₂ were the most effective. A complete LNCaP experiment compared the same series of oligos also in the presence of LD₅₀ Cytoxan. Each oligo combined with Cytoxan significantly inhibited: MR₁ by 49.0, MR₂ by 50.0, MR₄ by 53.0; MR₁₂ by 52.0; MR₂₁ by 58.6, MR₂₄ by 53.9 and MR₄₂ by 58.0%.     

Enhanced expression of bcl-2 following antisense oligonucleotide mediated growth factor deprivation

Although the role of bcl-2 in apoptosis has been described, its involvement in prostate cancer (CAP) progression is less well understood, but thought to be involved with the transition of CAP from androgen-sensitivity to androgen-independence, where its expression is augmented following androgen ablation. For treating these recurrent androgen-independent tumors, following hormone treatment failure, a new tier of therapy based upon growth factor deprivation has been suggested, implemented by antisense oligonucleotides (oligos) directed against mRNA encoding a critical growth regulatory autocrine loop (comprised of transforming growth factor-α (TGF-α) and its binding site, the epidermal growth factor receptor (EGFR). To determine whether oligo-induced growth factor deprivation therapy similarly enhanced expression of bcl-2 (as follows androgen deprivation) human prostate cancer derived PC-3 cells were treatedin vitro with oligos directed against TGF-α (MR-1) and/or EGFR (MR-2). After 5 days of treatment cells were immunochemically stained for human bcl-2. In similar experiments, cells were treated for 3 days prior to extraction of proteins, Western blot analysis, photography and computer evaluation of protein density by SigmaScan software. Immunostained cells treated with oligos directed against mRNA encoding TGF-α (MR-1) either alone or in combination with that directed against EGFR (MR-2) had increased bcl-2 expression (+3 to +5). In addition, the intensity of Western blots scanned for bcl-2 expression were 19%, 32% and 30% greater in cells treated with oligos directed against TGF-α, EGFR or their combination, respectively. We conclude that enhanced bcl-2 expression followed antisense oligo induced growth factor deprivation. This result is similar to that found upon androgen deprivation therapy, and also demonstrates additional biologic activity of this new class of molecular therapeutic agents.     

Treatment of prostate and breast tumors employing mono- and bi-specific antisense oligonucleotides targeting apoptosis inhibitory proteins clusterin and bcl-2

Antisense oligonucleotides (oligos) have demonstrated their efficacy in inhibiting the growth of prostate and breast tumor cells. Previous studies employed first generation, phosphorothioated, cDNA oligos synthesized complimentary to mRNA encoding transforming growth factor-alpha (TGF-α), epidermal growth factor receptor (EGFR), the anti-apoptosis protein bcl-2, and the androgen receptor (AR). In an effort to construct oligos with greater than one mRNA binding site, bi-specifics have been developed which target combinations of the above proteins, and these have been shown at least as effective as the mono-specific oligos from which their sequences were derived. While all bi-specifics have inhibitory effects, which can be enhanced by the combined administration of an additional chemotherapeutic agent, those bi-specifics which target bcl-2 and EGFR were reported to be the most effective. The experiments presented here are an effort to evaluate a new group of bi-specifics whose targets include the chaperone protein clusterin, whose expression is up regulated in many tumors and activity is known to inhibit apoptosis. Of particular interest were those bi-specifics constructed to target both clusterin and bcl-2 (also an apoptosis inhibitory protein). Cell lines targeted included both prostate LNCaP and PC-3, as well as the breast derived MCF-7. In order to identify agents which enhance oligo activity, but contribute less toxicity, oligos were tested both alone and in combination with either the immune inhibitor Rapamycin, or the chemotherapeutic (and more toxic) Taxol. Results indicate that bi-specifics targeting clusterin are statistically effective, and are similarly enhanced by Rapamycin, or Taxol. When bi-specifics including clusterin as a target, were tested against LNCaP and MCF-7 cells, the level of activity was intermediate between that of the mono-specific compounds tested separately. In experiments which compared both, bi-specifics which included a target for clusterin had inhibitory activity similar to the previously described bi-specifics directed towards bcl-2 and EGFR.     

Molecular determinants of cell death induction following adenovirus-mediated gene transfer of wild-type p53 in prostate cancer cells

Adenoviral vectors expressing wild-type p53 (Ad-p53) induce apoptosis in different types of cancer cells. The therapeutic utility of Ad-p53 is now being evaluated in prostate-cancer patients. Bcl-2 is frequently expressed by prostate-cancer cells and has previously been shown to inhibit p53-mediated cell death following genotoxic stress. We studied the impact of bcl-2 on Ad-p53-induced cell death in human prostate-cancer cells. Human prostate-cancer cell lines LNCaP (p53 wt) and PC3 (p53 mut) were stably transfected with bcl-2. After p53 transduction, cell viability, apoptosis induction and modulation of specific apoptosis-regulatory proteins were assessed. LNCaP vector control and bcl-2-expressing cells underwent similar decreases in viability associated with apoptosis induction following Ad-p53 infection. Increased bcl-2 expression provided significant protection to PC3 cells transduced with Ad-p53. These findings are correlated with modulations in bax, bcl-2, bcl-x(L) and p21 protein levels. These data suggest that Ad-p53 may be useful in the treatment of some prostate cancers.     

Decreased expression of bcl-2 in moderate and severe oral epithelia dysplasias

The bcl-2 gene family plays an important role in regulation of apoptosis. We previously reported loss of bcl-2 in peripheries of well and moderately differentiated oral squamous cell carcinoma tumour islands. We aimed to determine bcl-2 and bax gene expression in oral epithelial dysplasias (OED) in relation to apoptosis and proliferation. Samples of normal oral epithelium (OE, n=7), focal epithelial hyperplasia (n=9), mild (n=9), moderate (n=8) and severe (n=18) OED were evaluated by immunohistochemistry, the TUNEL method and in situ hybridisation for bcl-2 and bax mRNA. bcl-2 mRNA and protein were markedly decreased in the basal parts of moderate and severe OED compared with the basal layer of OE (P<0.001) and correlated with a 3-4 fold increase in apoptosis and increased proliferation. bax mRNA and protein were not significantly altered in normal, hyperplastic and dysplastic epithelia. From OE to severe OED, there was an inverse relationship between the bcl-2/bax ratio and apoptosis. Our results indicate that suppression of bcl-2 may have a role in oral tumourigenesis.     

Mechanistic studies of the effects of the retinoid N-(4-hydroxyphenyl)retinamide on prostate cancer cell growth and apoptosis

To explore the mechanisms underlying the chemopreventive effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in prostate cancer, we evaluated the anti-proliferative and apoptosis-inducing effects of 4-HPR in the androgen-sensitive human prostate cancer cell line LNCaP. 4-HPR decreased the number of viable LNCaP cells (as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) in a dose-dependent manner. Although 4-HPR exerted a modest G1 cell-cycle block (as determined by flow cytometry), its effect on reduced cell number appeared to result primarily from induction of apoptosis (as measured by an enzyme-linked immunosorbent assay and flow-cytometric assays). The mitogenic effects of R1881, a non-metabolizable androgen that potently induces LNCaP cell proliferation, was completely blocked by greater than 0.5 microM 4-HPR. Furthermore, increasing the R1881 concentration in the presence of 2.0 microM 4-HPR increased apoptotic cell death. 4-HPR decreased prostate-specific antigen (PSA) protein levels in conditioned medium and decreased PSA mRNA expression. 4-HPR also decreased the ratio of bcl-2 to bax mRNA expression in LNCaP cells by approximately 45%, indicating that the apoptotic effects of 4-HPR may be mediated, at least in part, by alterations in the bcl-2/bax-regulated apoptotic pathway. N-acetylcysteine (4 mM) completely blocked the anti-proliferative and apoptotic-inducing effects of 4-HPR, suggesting that an oxidative mechanism may be involved. We concluded that (i) 4-HPR exerts growth-suppressive and apoptotic effects on LNCaP cells, (ii) 4-HPR can interact with androgen to suppress proliferation and induce apoptosis, (iii) the apoptotic effects of 4-HPR may be mediated in part by the bcl-2/bax pathway, and (iv) a pro-oxidant mechanism may contribute to the anti-proliferative and apoptotic-inducing effects of 4-HPR.     

Altered Nuclear Localization of Bax Protein in BCNU-resistant Glioma Cells

To investigate the role of apoptosis suppression in glioma chemotherapy resistance, protein levels and subcellular localization of bcl-2 family members were investigated in 2 pairs of sensitive cell lines and their in vitro generated resistant derivatives. The alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), induced apoptosis in both sensitive cell strains and apoptosis was suppressed in both resistant derivatives. Both resistant cell lines contained altered regulation of a bcl-2 related protein consistent with the suppression of apoptosis. Independent of which bcl-2 family member was dysregulated, resistance was associated with altered regulation in the subcellular localization of bax protein. Following BCNU treatment, bax accumulated in nucleoli and a nuclei containing fraction of sensitive cells but not their resistant derivatives. Nuclear accumulation was an early event in apotosis induction. These data indicates altered subcellular localization of bax may play a role in resistance. In addition, the association between an early, nucleolar localization of bax and the induction of apoptosis suggests that localization of bax to nucleoli may play a role in apoptosis-induction of glioma cells.     

幽门螺杆菌感染性胃炎细胞凋亡与bcl-2/bax表达的意义

目的：探讨幽门螺杆菌ＨＰ引起胃粘膜上皮细胞凋亡和增殖发迹及ＨＰ致癌作用的可能机制。方法：采用免疫组织化学方法及脱氧核糖核酸末端转移酶介导的ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ）技术,对ＨＰ阳性和ＨＰ阴性者胃粘膜上皮中增殖细胞,凋亡细胞进行原位观察和比较,同时检测ｂｃ１－２／ｂａｘ蛋白表达状态。结果：ＨＰ阳性者胃粘膜上皮细胞增殖指数和凋亡指数显著高于ＨＰ阴性者（Ｐ〈０．０１）。ｂａｘ蛋白表达阳性率在ＨＰ     

三氧化二砷诱导雌激素受体阴性乳腺癌细胞株MDA-MB-435s凋亡和bcl-2、bax表达关系的研究

目的 观察不同浓度三氧化二砷(As2O3)诱导人乳腺癌细胞系MDA-MB-435s凋亡的情况及对bcl-2和bax表达的影响,初步探讨了其诱导凋亡的途径.方法 不同浓度As2O3体外作用于MDA-MB-435s细胞24h后,采用TUNEL染色法和DNA梯状电泳法检测细胞凋亡情况,应用免疫组化法检测细胞中bcl-2和bax蛋白表达情况.结果 As2O3作用后,DNA琼脂糖凝胶电泳法呈现凋亡DNA梯状条带;As2O3作用组的凋亡指数明显高于阴性对照组(P＜0.05);bcl-2表达下调,bax表达上调.结论 As2O3能诱导MDA-MB-435s细胞凋亡,上调bcl-2蛋白表达、下调bax蛋白表达是其诱导凋亡的途径之一.     

Expression of bcl-2, bax,and bcl-XL proteins in azoxymethane-induced rat colonic adenocarcinomas

Using western blotting and immunochemical analysis, we investigated alterations in the expression of the apoptosis-related proteins bcl-2, bax, and bcl-X in colonic adenocarcinomas induced by subcutaneous injection of azoxymethane (AOM) (15 mg/kg body weight weekly for 2 wk) into male Sprague-Dawley rats. Expression of the apoptosis-repressor bcl-2 in the colonic tumors was significantly weaker (0.6-fold) than that in adjacent non-neoplastic mucosa. The expression of bax protein, an apoptosis accelerator, was significantly stronger (7.33-fold) in all the tumors than in the non-tumoral mucosa. bcl-X_L protein, which functions as a repressor of apoptosis, was significantly upregulated (3.23-fold) in all the tumors when compared with the non-neoplastic mucosa. There was no significant difference between the expression of these proteins in the non-neoplastic mucosa of the AOM-treated rats and in the normal mucosa of saline-treated control rats. As determined by immunohistochemical analysis, the tumor cells had more bax and bcl-X protein. These findings indicate that the regulation of the apoptosis-related proteins bcl-2, bax, and bcl-X_L was altered in the AOM-induced colonic neoplastic tissue. In terms of resistance to apoptosis, elevated levels bcl-X_L protein may have considerable meaning in this experimental model as well as in human colorectal cancer. Mol. Carcinog. 19:25–30, 1997. © 1997 Wiley-Liss, Inc.     

Spontaneous apoptosis in laryngeal squamous cell carcinoma is independent of bcl-2 and bax protein expression

BACKGROUND: bcl-2 and bax genes are known to be involved in the control of apoptotic cell death, an important mechanism of growth regulation that influences the biologic behavior of tumors. The aim of the current study was to investigate the relationship of bcl-2 and bax expression to the rate of spontaneous apoptosis in laryngeal carcinomas, and to assess its relations to clinicopathologic features of tumors. METHODS: Immunohistochemical analyses for bcl-2 and bax were performed on paraffin embedded tissue sections from 134 primary laryngeal squamous cell carcinomas. To visualize apoptotic cells, the nick end labeling method was used. The proliferative activity of tumors was analyzed by determination of mitotic indices. RESULTS: bcl-2 immunoreactivity was positively correlated with tumor grade (P < 0.00001), high T category (P < 0.02), metastatic involvement of cervical lymph nodes (P < 0.003), and supraglottic or subglottic location of primary tumors (P < 0.00005). An inverse relation was found between bcl-2 and bax expression (P < 0.004). The frequency of spontaneous apoptosis was closely associated with mitotic activity (P < 0.0004) but appeared to be unrelated to protein levels of bcl-2 or bax as well as to bcl-2:bax ratios. CONCLUSIONS: The results of this study point to the significance of cell proliferation as a major determinant of the rate of spontaneous apoptosis in laryngeal carcinomas. The bcl-2:bax expression ratio obviously does not affect the incidence of apoptosis, but it may be considered as a marker of disease progression and poor prognosis.     

p53-Independent induction of apoptosis in human melanoma cells by a bcl-2/bcl-xL bispecific antisense oligonucleotide.

Mutations in the p53 tumor suppressor gene are implicated in defective apoptotic response of tumors to genotoxic damage and, thus, are major determinants of resistance to a variety of anticancer agents. Because even melanomas harboring wild-type (wt) p53 show an abnormal response to radiation and p53 mutations occur late during melanoma progression, we investigated whether the effect of the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 is dependent on the p53 status in human C8161 melanoma cells. Upon treatment with oligonucleotide 4625, p53-mut C8161 cells showed earlier DNA damage, which occurred concomitantly with the reduction of bcl-2 and bcl-xL expression and the increase in the expression of proapoptotic bax. Loss of cell viability, bcl-2 down-regulation, and poly(ADP-ribose) polymerase cleavage, indicative of apoptosis, also occurred in wt p53 C8161 cells on treatment with oligonucleotide 4625. These effects, however, were mediated by strong induction of p53 without changes in p21 WAF1 expression in wt p53 cells, whereas a 70% decrease in p21 WAF1 expression was observed in mut p53 cells. In contrast to many other anticancer agents to which the apoptotic response is decreased because of p53 mutations, our data suggest that the bcl-2/bcl-xL bispecific antisense oligonucleotide 4625 effectively induces p53-independent apoptosis in human C8161 melanoma cells.