Approach to PTHrP gene therapy for oral cancer

在以往的临床免疫组织化学研究中，作者发现甲状旁腺激素相关蛋白(PTHrP)在口腔鳞状细胞癌中表达，其定位与角化程度较高和组织学恶性程度较高的区域相对应。本研究旨在探讨甲状旁腺素rP对口腔鳞状细胞增殖和分化的影响。我们使用了来自舌癌的鳞状癌细胞系SCC-25和来自人牙龈组织的永生化角化细胞系NDUS-1。将PTHrP(1-34)、(34-53)和(107-139)片段加入培养基中。据报道，PTHrP的每个片段都有不同的生理功能，如调节平滑肌张力、跨皮细胞钙转运以及组织和器官的发育、分化和增殖。为了评估细胞的增殖，我们计算了细胞的数量。采用免疫荧光染色技术检测这些片段对分化的影响，包括总蛋白、细胞角蛋白(CK)10、CK14、CK5、CK6和CK18。我们还评估了细胞内cAMP浓度。经片段刺激后，两种细胞系的细胞数量和免疫荧光染色均未见明显变化。加入这些片段后，两株细胞内cAMP浓度均无明显变化。在基因芯片研究中，PTHrP片段(1-34)没有增加NDUS-1中CK的表达。虽然在反义RNA研究中发现PTHrP影响角质形成细胞的分化，但我们没有观察到外源加入PTHrP对细胞分化和增殖的影响。这些片段在口腔鳞状细胞中的内源性功能有待进一步研究。此外，我们还利用基因芯片研究了PTHrP的体外作用。PTHrP不能降低影响细胞增殖的基因表达。

In previous clinical immunohistochemical studies, the authors found that parathyroid hormone-related protein (PTHrP) was expressed in oral squamous cell carcinoma in which the localization corresponded to regions of higher keratinization and tower grade of histologic malignancy. The purpose of this study was to examine the effect of PTHrP on proliferation and differentiation of oral squamous cells. We used a squamous carcinoma cell line, SCC-25, derived from tongue cancer, and an immortalized keratino cyte cell line, NDUSD-1, derived from human gingival tissue. PTHrP (1-34), (34-53), and (107-139) fragments were added to the culture medium. It has been reported that each of PTHrP fragments has different physiologic functions, such as regulation of smooth muscle tone, transepithelial calcium transport, and tissue and organ development, differentiation, and proliferation. For assessment of proliferation, we counted the number of cells. The ability of the fragments to affect differentiation was evaluated by using immunofluorescent staining with involucrin, cytokeratin (CK)10, CK14, CK5, CK6, and CK18. We also assessed intracellular cAMP concentration. No changes in cell number or immunofluorescence staining were evident in either cell line following stimulation with the fragments. The concentration of cAMP did not change in either cell line after adding the fragments. PTHrP fragment (1-34) did not increase CK expressions in NDUSD-1 in a cDNA microarray study. Although it has been reported that PTHrP affects differentiation of keratinocytes on antisense RNA study, we did not observe any influence of exogenously added PTHrP on cell differentiation and proliferation in the examined cell lines. The endogenous function of the fragments in oral squamous cells requires further study. Furthermore we examined in vitro effects of PTHrP by using cDNA array study. PTHrP could not reduce gene expression affecting cell proliferation.