Growth-inhibitory effects of CD40 ligand (CD154) and its endogenous expression in human breast cancer.
Growth-inhibitory effects of CD40 ligand (CD154) and its endogenous expression in human breast cancer.
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发表时间：
2001-03
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通讯作者：
A. Tong;Maria H. Papayoti;G. Netto;D. Armstrong;G. Ordonez;J. Lawson;M. Stone
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作者：
A. Tong;Maria H. Papayoti;G. Netto;D. Armstrong;G. Ordonez;J. Lawson;M. Stone
CD40结合在正常和恶性B细胞中产生多方面的生长信号，而其在上皮癌中的生理作用尚不清楚。我们使用CD40+ （T47D和BT-20）和CD40阴性（MCF-7, ZR-75-1）细胞系，通过流式细胞分析、免疫组织化学和逆转录- pcr检测了CD40结扎在人乳腺癌细胞中的生长结果。用可溶性重组CD40配体（CD40L）分子gp39或CD40L-三聚体处理后，BT-20和T47D细胞对[3H]胸苷的摄取可显著降低40%，但不影响CD40阴性的MCF-7或ZR-75-1细胞的生长。同样，与组成表达膜CD40L的转染小鼠L细胞（55.0 +/- 8.9%,P < 0.001）或与来自三种不同hla错配供者的多聚甲醛固定的CD3+ CD40L+ pbl共孵育（39.7 +/- 3.7%,P < 0.01）后，观察到显著的生长抑制。未转染的L细胞和不表达cd40l的淋巴细胞不产生明显的生长抑制。CD40L的体内抗肿瘤作用采用sc严重联合免疫缺陷-hu异种移植模型。用两种不同的可溶性重组CD40L构建体（CD40L和gp39）预处理产生了相似的异种移植物生长抑制作用[分别抑制67 +/- 24% （n = 4）和65 +/- 14% (n = 8)]，通过与CD40L中和抗体LL48共同处理，这种抑制作用被逆转。体外分析表明，cd40l诱导的生长抑制伴随着凋亡事件，包括细胞收缩、圆圆和从粘附的T47D培养单层脱落。gp39处理的T47D和BT-20细胞分别有31%和27%发生凋亡，而Fas激动抗体CH-11处理的T47D和BT-20细胞分别有56%和65%发生凋亡。在T47D和BT-20细胞中观察到促凋亡蛋白Bax的上调，表明该Bcl-2家族成员可能参与了这种生长抑制作用。为了探讨CD40L-CD40相互作用的临床相关性，我们进行了回顾性免疫组织化学分析，以表征乳腺癌患者活检中CD40-和cd40l的原位表达。浸润性导管癌（5例中有5例）、小叶癌（4例中有4例）、原位癌（6例中有6例）和黏液癌（1例）均表达CD40。在大多数浸润性导管癌（5例中有3例）、小叶癌（4例中有3例）和原位癌（6例中有4例）中，不同比例的肿瘤细胞也表达CD40L，这是通过免疫组织化学确定的，并通过RT-PCR检测CD40L信息，仅在CD40L阳性染色的病例中得到验证。浸润性癌和原位癌的浸润性单核细胞表达CD40（10例中有10例），但CD40L表达较少（浸润性小叶癌1例，原位癌2例）。我们的发现表明CD40信号通路在人乳腺癌细胞中是活跃的。然而，来自原发肿瘤组织的肿瘤浸润淋巴细胞通过CD40L-CD40环直接调节肿瘤生长的能力可能有限。
CD40 binding produces multifaceted growth signals in normal and malignant B cells, whereas its physiological role is less well characterized in epithelial cancers. We examined the growth outcome of CD40 ligation in human breast cancer cells, using CD40+ (T47D and BT-20) and CD40-negative (MCF-7, ZR-75-1) cell lines as defined by flow cytometric analysis, immunohistochemistry, and reverse transcription-PCR. Treatment with the soluble recombinant CD40 ligand (CD40L) molecules gp39 or CD40L-trimer significantly reduced [3H]thymidine uptake in BT-20 and T47D cells by up to 40%, but did not affect the growth of CD40-negative MCF-7 or ZR-75-1 cells. Similarly, significant growth inhibition was observed after co-incubation with CD40L-transfected murine L cells (55.0 +/- 8.9%, P < 0.001) that express membrane CD40L constitutively, or with paraformaldehyde-fixed, CD3+ CD40L+ PBLs from three different HLA-mismatched donors (39.7 +/- 3.7%, P < 0.01). Untransfected L cells and non-CD40L-expressing lymphocytes did not produce significant growth inhibition. The in vivo antitumorigenic effects of CD40L were examined using a s.c. severe combined immunodeficient-hu xenograft model. Pretreatment with two different soluble recombinant CD40L constructs (CD40L and gp39) produced similar xenograft growth-inhibitory effects [67 +/- 24% (n = 4), and 65 +/- 14% (n = 8) inhibition, respectively], which were reversed by co-treatment with the CD40L-neutralizing antibody LL48. In vitro analysis indicated that CD40L-induced growth inhibition was accompanied by apoptotic events including cell shrinkage, rounding, and detachment from the adherent T47D culture monolayer. Thirty-one and 27% of gp39-treated T47D and BT-20 cells underwent apoptosis, respectively, as compared with 56 and 65% from the same cell lines after treatment with the Fas agonistic antibody CH-11. An up-regulation of the proapoptotic protein Bax in T47D and BT-20 cells was observed, which indicated that this Bcl-2 family member may contribute to this growth-inhibitory effect. To explore the clinical relevance of CD40L-CD40 interaction, retrospective immunohistochemical analysis was carried to characterize in situ CD40- and CD40L-expression in breast cancer patient biopsies. All of the infiltrating ductal (5 of 5 cases tested) and lobular (4 of 4 cases) breast carcinomas, carcinomas in situ (6 of 6 cases), and mucinous carcinoma tested (1 case) expressed CD40. Varying proportions of tumor cells also expressed CD40L in the majority of infiltrating ductal (3 of 5 cases) and lobular (3 of 4 cases) carcinomas, and carcinomas in situ (4 of 6 cases), as determined by immunohistochemistry and validated by RT-PCR detection of the CD40L message in only CD40L positive-staining cases. Tumor infiltrating mononuclear cells from infiltrating carcinomas and carcinomas in situ expressed CD40 (10 of 10 cases), but less commonly CD40L (1 case of infiltrating lobular carcinoma, 2 cases of carcinoma in situ). Our findings indicate that the CD40 signaling pathway is active in human breast carcinoma cells. However, tumor-infiltrating lymphocytes from primary tumor tissues may be limited in their capacity to directly modulate tumor growth through the CD40L-CD40 loop.