不同途径免疫的AFP基因修饰DC瘤苗体内抗肿瘤效应的研究

目的:评价bcl-2反义寡核苷酸对肾细胞癌细胞Bcl-2蛋白表达抑制及诱导凋亡作用.方法:合成与已知人类基因无同源性的bcl-2反义寡核苷酸(AS1与翻译起始端互补,AS2与编码区互补),以阳离子脂质体Lipofectin为转染载体,MTS比色实验测定细胞活率,反转录-聚合酶链反应(RT-PCR)测定bcl-2 mRNA的表达,Western杂交法测定Bcl-2蛋白表达,碘化丙啶(PI)染色流式细胞术测定凋亡细胞.结果:所测定的5个肾癌细胞株均有稳定的bcl-2 mRNA表达;转染的反义寡核苷酸对ACHN细胞Bcl-2蛋白的表达有明显抑制作用,而正义寡核苷酸无明显影响,AS2的抑制作用大于AS1;反义寡核苷酸可诱导ACHN细胞的凋亡,AS1和AS2的诱导率分别为32.1%和43.2%.结论:bcl-2反义寡核苷酸可抑制肾癌细胞Bcl-2蛋白的表达,并进而诱导细胞的调亡.     

Preclinical studies of Apogossypolone: a new nonpeptidic pan small-molecule inhibitor of Bcl-2, Bcl-X<Subscript>L</Subscript> and Mcl-1 proteins in Follicular Small Cleaved Cell Lymphoma model

Elevated expression of anti-apoptotic Bcl-2 family proteins have been linked to a poor survival rate of patients with Follicular Lymphoma (FL). This prompted us to evaluate a very potent non-peptidic Small-Molecule Inhibitor (SMI) targeting Bcl-2 family proteins, Apogossypolone (ApoG2) using follicular small cleaved cell lymphoma cell line (WSU-FSCCL) and cell isolated from lymphoma patients. ApoG2 inhibited the growth of WSU-FSCCL significantly with a 50% growth inhibition of cells (IC₅₀) of 109 nM and decreased cell number of fresh lymphoma cells. ApoG2 activated caspases-9, -3, and -8, and the cleavage of Poly (ADP-ribose) polymerase (PARP) and Apoptosis Inducing Factor (AIF). In the WSU-FSCCL-SCID xenograft model, ApoG2 showed a significant anti-lymphoma effect, with %ILS of 84% in the intravenous and 63% in intraperitoneal treated mice. These studies suggest that ApoG2 can be an effective therapeutic agent against FL.     

Eradication of human non-Hodgkin's lymphoma in SCID mice by BCL-2 antisense oligonucleotides combined with low-dose cyclophosphamide.

Cancers overexpressing Bcl-2 protein, which prevents programmed cell death (apoptosis), are less sensitive to stresses that produce cellular damage, including chemotherapy. If the level of Bcl-2 protein can be reduced sufficiently using antisense oligonucleotides (ASOs) targeting the gene message, then cytotoxic agents may be rendered more effective in eliminating disease and increasing cure rate. Preclinical studies in SCID mice bearing Bcl-2 overexpressing systemic human B-cell lymphoma (DoHH2) were undertaken to support development of a clinical trial. These data confirm that a combination of an ASO (5 mg/kg) targeting bcl-2 and a low dose of cyclophosphamide (35 mg/kg) was an effective strategy, leading to the eradication of the DoHH2 cells in vivo and cure of the animals. When mice deficient in natural killer cell activity were treated with an ASO, similar results were observed, suggesting that ASO stimulation of the host immune system was not a significant factor in elimination of lymphoma cells. These studies indicate that therapeutic strategies involving the use of an ASO targeting bcl-2 in combination with a cytotoxic agent may improve clinical outcomes.     

Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model.

Bcl-2 has emerged as a critical regulator of apoptosis in a variety of cell systems and is up-regulated during progression to androgen independence in prostate cancer cells. The objectives of this study were to characterize changes in Bcl-2 after androgen withdrawal and during progression to androgen independence in the human prostate LNCaP tumor model and determine whether adjuvant use of antisense Bcl-2 oligodeoxynucleotides (ODNs) with androgen ablation delays progression to androgen independence. Bcl-2 expression in LNCaP cells is down-regulated to undetectable levels by androgen in vitro and up-regulated after castration in vivo. Antisense Bcl-2 ODN treatment reduced LNCaP cell Bcl-2 messenger RNA and protein levels by >90% in a sequence-specific and dose-dependent manner at concentrations >50 nM. Bcl-2 mRNA levels returned to pretreatment levels by 48 h after discontinuing treatment. Athymic male mice bearing SQ LNCaP tumors were castrated and injected i.p. with 12.5 mg/kg/day with two-base mismatch ODN control, reverse polarity ODN control, or antisense Bcl-2 ODN. Tumor volume in control mice gradually increased 5-fold (range, 3-6) by 12 weeks after castration compared to a 10-50% decrease in precastrate tumor volume in mice treated with antisense Bcl-2 ODN. Changes in serum PSA paralleled changes in tumor volume, increasing 4-fold faster above nadir in controls than in mice treated with antisense Bcl-2 ODN. After decreasing 70% by 1 week after castration, PSA increased 1.6-fold above precastrate levels by 11 weeks in controls while staying 30% below precastrate levels in antisense-treated mice. In a second group of experiments, LNCaP tumor growth and serum PSA levels were 90% lower (P<0.01) in mice treated with antisense Bcl-2 ODN compared with mismatch or reverse polarity ODN controls. These results support the hypothesis that Bcl-2 helps mediate progression to androgen independence and is an appropriate target for antisense therapy.     

Inhibition of progression to androgen-independence by combined adjuvant treatment with antisense BCL-XL and antisense Bcl-2 oligonucleotides plus taxol after castration in the Shionogi tumor model

We have reported that antisense Bcl-2 oligodeoxynucleotide (ODN) delays progression to androgen independence in the androgen-dependent (AD) mouse Shionogi tumor model. Here, we characterize changes in bcl-xL, another important anti-apoptotic gene, and test the efficacy of adjuvant antisense Bcl-xL ODN therapy either alone or in combination with antisense Bcl-2 ODN and chemotherapy after castration in the Shionogi tumor model. Bcl-xL mRNA levels increased up to 3-fold postcastration and remained 1. 5-fold higher in androgen-independent (AI) recurrent tumors compared with AD tumors before castration. Treatment of Shionogi cells with antisense Bcl-xL ODN inhibited Bcl-xL expression in a dose-dependent and sequence-specific manner. Systemic administration of antisense Bcl-xL ODN in mice bearing Shionogi tumors after castration delayed emergence of AI recurrent tumors. We then examined whether combined adjuvant antisense Bcl-xL and/or Bcl-2 ODNs plus taxol (paclitaxel) therapy further delays time to AI progression. Combined treatment of Shionogi cells with antisense Bcl-xL and Bcl-2 ODNs significantly enhanced taxol chemosensitivity compared with either agent alone, reducing the IC(50) of taxol by more than 1 log. Apoptotic DNA laddering and cleavage of poly(ADP-ribose) polymerase were more substantial after treatment with combined antisense Bcl-2 and Bcl-xL ODNs plus taxol than that with either 2 agents. Adjuvant administration of antisense Bcl-xL and Bcl-2 ODNs plus micellar taxol resulted in a significantly delayed time to AI recurrence compared with administration of either 2 agents. Our findings suggest that Bcl-xL represents a suitable molecular target for antisense ODN strategy and illustrate the potential additive effects of multi-target pharmacology for cancer therapy.     

Chemosensitization of myeloma plasma cells by an antisense-mediated downregulation of Bcl-2 protein.

An antisense oligodeoxynucleotide (ODN) complementary to the first six codons of the Bcl-2 mRNA, G3139 (oblimersen sodium; Genasense), has been shown to downregulate Bcl-2 and produce responses in a variety of malignancies including drug-resistant lymphoma. Incubation of ex vivo purified plasma cells from patients with multiple myeloma (MM) with carboxyfluorescein (FAM)-labeled antisense ODNs resulted in a time- and dose-dependent uptake in the cytoplasm and nucleus. No major differences in uptake of Bcl-2 antisense ODNs were observed among patients' samples. Incubation of purified myeloma plasma cells with G3139, but not solvent or reverse polarity control ODNs, resulted in a reduction (>75%) of Bcl-2 mRNA levels after 2 and 4 days, as measured by Real-Time PCR. Treatment with G3139 led to a sequence-specific reduction of Bcl-2 protein levels within 4 days of exposure in 10 out of 11 clinical samples from patients with chemosensitive and multidrug-resistant disease, without significant reduction of alpha-Actin, Bax, Bcl-XL, or Mcl-1 proteins. This resulted in a significantly enhanced sensitivity of the myeloma tumor cells to dexamethasone or doxorubicin-induced apoptosis. G3139 can consistently enter myeloma cells, downregulate the expression of Bcl-2, and enhance the efficacy of myeloma therapy. These data support further clinical evaluation of G3139 therapy in multiple myeloma.     

Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucleotide therapy in patients with non-Hodgkin's lymphoma.

PURPOSE: To evaluate the pharmacokinetics and toxicity of an antisense oligonucleotide targeting bcl-2 in patients with non-Hodgkin's lymphoma (NHL) and to determine efficacy using clinical and biologic end points. PATIENTS AND METHODS: Twenty-one patients with Bcl-2-positive relapsed NHL received a 14-day subcutaneous infusion of G3139, an 18-mer phosphorothioate oligonucleotide complementary to the first six codons of the bcl-2 open reading frame. Plasma pharmacokinetics were measured by anion exchange high-performance liquid chromatography. Response was assessed by computed tomography. Changes in Bcl-2 expression were measured by fluorescence-activated cell sorting of patients' tumor samples. RESULTS: Eight cohorts of patients received doses between 4. 6 and 195.8 mg/m(2)/d. No significant systemic toxicity was seen at doses up to 110.4 mg/m(2)/d. All patients displayed skin inflammation at the subcutaneous infusion site. Dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. The maximum-tolerated dose was 147.2 mg/m(2)/d. Plasma levels of G3139 equivalent to the efficacious plasma concentration in in vivo models were produced with doses above 36.8 mg/m(2)/d. Plasma levels associated with dose-limiting toxicity were greater than 4 microg/mL. By standard criteria, there was one complete response, 2 minor responses, nine cases of stable disease, and nine cases of progressive disease. Bcl-2 protein was reduced in seven of 16 assessable patients. This reduction occurred in tumor cells derived from lymph nodes in two patients and from peripheral blood or bone marrow mononuclear cell populations in the remaining five patients. CONCLUSION: Bcl-2 antisense therapy is feasible and shows potential for antitumor activity in NHL. Downregulation of Bcl-2 protein suggests a specific antisense mechanism.     

Therapeutic targeting of oncogenic transforming growth factor-β1 signaling by antisense oligonucleotides in oral squamous cell carcinoma

The transforming growth factor-β1 (TGF-β1) signaling pathway is important in human oral squamous cell carcinoma (OSCC). Accordingly, the aims of this study were to evaluate the effect of antisense TGF-β1 oligonucleotides (ODNs) on OSCC in cell culture and in a xenograft model, as well as to evaluate any effects ODNs have on proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase-2 (MMP-2) expression in the xenograft model. We performed real-time cell electronic sensing (RT-CES) to determine the effect of antisense TGF-β1 ODNs on SCC-9?cell growth. To examine the in?vivo effect of antisense TGF-β1 ODN therapy, SCC-9?cells were grafted into nude mice. Antisense ODNs were injected into the mass daily. Tumor size, body weight and duration of survival were assessed daily. Specimens from the main mass were used for immunohistochemical staining to analyze PCNA and MMP-2 expression. In?vitro treatment with antisense TGF-β1 ODNs decreased TGF-β1 expression and growth of SCC-9?cells. In the xenograft model, the antisense TGF-β1 ODN group exhibited a significantly decreased tumor growth rate compared to the control, which received Dulbecco's modified Eagle's medium (DMEM) (P=0.022). However, mean survival time and body weights were not significantly different between the groups (P>0.05). Immunohistochemistry showed that tumors from animals that received antisense TGF-β1 ODNs had significantly lower expression levels of PCNA and MMP-2 compared to tumors from animals in the DMEM group (P<0.05). In conclusion, antisense TGF-β1 ODN therapy significantly inhibits tumor growth compared to controls, however, there are no significant differences between groups with respect to changes in body weight.     

BCL-2 gene in lymphocytic malignancy

In t(14;18) (q32;q21) lymphomas, bcl-2 gene is activated by the juxtaposition of immunoglobulin (Ig) gene. The fused bcl-2-Ig gene generates chimeric mRNAs which consist of bcl-2 at 5' portion and Ig at 3' portion. Chimeric mRNA does not disrupt the bcl-2 coding frame of 239 amino acid polypeptide. Bcl-2-Ig transgenic mice demonstrated the extended B cell survival and the follicular lymphoproliferation, but they did not develop a malignancy until 25 weeks. Ten percent of them, however, developed malignant diffuse large-cell lymphomas after a long latency. Forty percent of these malignancies demonstrated the c-myc rearrangement, indicating that multiple step changes are required for malignant transformation in bcl-2 activated cells. Study on the bcl-2 gene rearrangement in Japanese B cell lymphoma and B-CLL revealed that 10 out of 32 cases of follicular lymphoma (31%), 5 out of 56 cases of diffuse lymphoma (9%) and 2 out of 30 cases of B-CLL (7%) were rearranged. Less frequency of B cell lymphoma, particularly follicular lymphoma in Japan might be partly due to the less bcl-2 involvement than in American cases. The ratio of bcl-2 involvement in B-CLL is not significantly different between Japan and U.S.A.. bcl-2 rearrangement at 5' promoter region is noted for Japanese B-CLL which was demonstrated for American cases. The clinical application of polymerase chain reaction for bcl-2 translocation was also discussed.     

Primary follicular lymphoma of the testis in childhood

BACKGROUND: Follicular lymphoma in childhood is rare. The authors present four unusual primary follicular lymphomas of the testis in children. METHODS: Tumor tissue was evaluated using light microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction (PCR) for immunoglobulin heavy chain (IgH) and bcl-2 gene rearrangements. Southern blot and immunohistochemical analyses were used to detect bcl-6 gene rearrangements and protein expression, respectively. RESULTS: Four young boys ranging in age from 3 to 10 years were diagnosed with Stage IE follicular large cell lymphoma (Grade 3). A B-cell phenotype was documented in all four cases; monoclonality was confirmed in three cases by demonstration of light chain restriction or clonal IgH gene rearrangement. None of the lymphomas expressed Bcl-2 or p53 protein, and bcl-2 gene rearrangements were not found in the three lymphomas studied. In contrast, Bcl-6 protein was expressed by all three lymphomas studied, and a bcl-6 gene rearrangement was detected in the one case analyzed by Southern blot. All four boys were treated by orchiectomy and combination chemotherapy and are alive with no evidence of disease 18-44 months following their initial diagnoses. CONCLUSIONS: Follicular lymphomas may rarely occur as primary testicular tumors in prepubertal boys and, when localized, appear to be associated with a favorable prognosis. In contrast to follicular lymphoma in adults, pediatric follicular lymphomas of the testis are usually of large cell type (Grade 3) and lack bcl-2 or p53 abnormalities. The identification, in one case, of a bcl-6 gene rearrangement suggests an alternate molecular pathogenesis for pediatric follicular lymphoma.     

Liposomal Targeting of Bcl-2 Antisense Oligonucleotides with Enhanced Stability into Human Myeloma Cell Lines

Cationic liposomes improve the delivery of antisense oligonucleotides (ODNs) into cells. However, there is marked variability in the cellular uptake of ODNs into different cell lines. We used liposomes containing dimethyloctadecylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE) to increase the delivery of phosphodiester ODNs into four different myeloma cell lines. The delivery by cationic liposomes increased the delivery of bcl-2 antisense ODNs by a factor of 9 to 45 as compared to plain ODNs. The stability of ODNs was increased with liposomes both in the culture medium and within the cells. Intact liposomal ODNs were detected inside the cells up to 24 hours with gel electrophoresis and phosphor imager analysis. Antisense ODNs had no effect on bcl-2 mRNA levels. Also the proliferation of myeloma cells remained unchanged during the 3-day incubation period. Our study shows that liposomal antisense ODNs targeting bcl-2 of human myeloma cells result in increased stability of ODNs with minimal toxicity. However, further modifications are needed to gain biological effects of antisense ODNs on human myeloma cells.     

Systemic Bcl-2 antisense oligodeoxynucleotide in combination with cisplatin cures EBV+ nasopharyngeal carcinoma xenografts in SCID mice

Nasopharyngeal carcinoma (NPC) is causally linked to Epstein-Barr virus (EBV), and the EBV oncoprotein, latent membrane protein 1 (LMP-1), is expressed in the majority of NPCs. LMP-1 upregulates antiapoptotic genes, including bcl-2, and Bcl-2 protein is overexpressed in NPC. Given the antiapoptotic and chemoprotective effects of Bcl-2, it represents a rational therapeutic target in NPC. We have investigated the antitumor and chemosensitizing effects of the Bcl-2 antisense oligodeoxynucleotide G3139 (oblimersen, Genasense) in NPC. For these studies, we used the C666-1 line, a stably infected NPC-derived line that co-expresses LMP-1 and Bcl-2. We have shown that G3139 treatment of C666-1 in vitro caused sequence-dependent suppression of Bcl-2 protein, inhibition of cell growth and enhanced sensitivity to cisplatin (CDDP), as measured by increased antiproliferative and apoptotic effects. In vivo, G3139 treatment (25 mg/kg every 3 days x 5 doses) delayed engraftment and significantly inhibited growth of established C666-1 xenografts in SCID mice compared to control oligo-treated animals. However, G3139 alone did not prevent engraftment or cure established tumors in any animals. In contrast, G3139 treatment (25 mg/kg every 3 days x 5 starting on day 7) in combination with CDDP (8 mg/kg on day 14) completely abrogated tumor engraftment in 80% of animals compared to CDDP (0%) or CDDP + control oligo (0%). When treatment was delayed until tumor was established, G3139 in combination with CDDP significantly inhibited tumor growth compared to CDDP or CDDP + control oligo, and cured 69% animals with established tumors. No animals treated with G3139, CDDP or CDDP + control oligo were cured. Tumor burden and response to treatment correlated with EBV DNA load in serum, measured by real-time PCR. Western blots of tumor extracts obtained during oligo treatment showed that Bcl-2 levels were significantly decreased in G3139-treated animals. Our studies have demonstrated that the Bcl-2 antisense oligodeoxynucleotide, G3139, has proapoptotic effects in C666-1, and in combination with CDDP, is curative in C666-1 NPC xenograft tumors in vivo. The sequence-dependency of these effects is consistent with an antisense mechanism. These studies suggest that Bcl-2 may represent a biologically relevant target for the development of novel combinatorial therapies for NPC.     

Growth of human lymphoma cells in SCID mice.

Two EBV-negative human lymphoma cell lines raised in this laboratory and peripheral blood cells of a patient with large cell lymphoma in leukemic phase were injected intravenously or intraperitoneally into C.B.17 SCID mice. One line (OCI-LY18) was derived from the pleural fluid of a patient with a large cell, immunoblastic malignant lymphoma. Cells of this line are of B cell origin and characterized by multiple rearrangements of the JH locus. The second line (OCI-LY17) was grown from the peripheral blood of a patient with a large cell lymphoma of T-cell phenotype which has characteristic rearrangement of the T-cell receptor beta chain. The cells directly obtained from a patient with large cell non-cleaved malignant lymphoma were of B-cell origin. Animals carrying OCI-LY18 developed large tumor masses within 6-8 weeks of inoculation. The tumors were detected in the intestine, mesentery, retroperitoneum, lymph nodes, spleen, lung and kidney. The masses resembled the primary tumor with respect to histological appearance and immunological phenotype. It was possible to generate secondary cell lines from the tumors found in the inoculated SCID mice. Injection of one of these secondary cell lines into SCID mice resulted in the rapid development of lymphoma and as few as 10 cells were sufficient to establish the disease in the inoculated animals. In contrast cells of OCI-LY17 produced small tumor aggregates that did not appear to progress over time and did not cause death of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of bcl-2 expression in normal and leukemic human B-cell differentiation.

Lack of apoptosis has been linked to prolonged survival of malignant B cells expressing bcl-2. The aim of the present study was to analyze the amount of bcl-2 protein expressed along normal human B-cell maturation and to establish the frequency of aberrant bcl-2 expression in B-cell malignancies. In normal bone marrow (n=11), bcl-2 expression obtained by quantitative multiparametric flow cytometry was highly variable: very low in both CD34(+) and CD34(-) B-cell precursors, high in mature B-lymphocytes and very high in plasma cells. Bcl-2 expression of mature B-lymphocytes from peripheral blood (n=10), spleen (n=8) and lymph node (n=5) was significantly higher (P<0.02) in CD23(-) as compared to CD23(+) B cells, independent of the type of tissue analyzed. Upon comparison with normal human B-cell maturation, bcl-2 expression in neoplastic B cells from 144 patients was found to be aberrant in 66% of the cases, usually corresponding to bcl-2 overexpression (63%). Follicular lymphoma (FL) carrying t(14;18) and MALT lymphoma were the only diagnostic groups constantly showing overexpression of bcl-2. Bcl-2 overexpression was also frequently found in precursor B-acute lymphoblastic leukemia (84%), typical (77%) and atypical (75%) B-cell chronic lymphocytic leukemia, prolymphocytic leukemia (two of three cases), mantle cell lymphoma (55%), but not in t(14;18)(-) FL, splenic marginal zone lymphoma, Burkitt lymphoma and multiple myeloma.