Mrowietz U, Schwenk U, Maune S, Bartels J, Küpper M, Fichtner I, Schröder J M, Schadendorf D
Department of Dermatology, Univeristy of Kiel, Germany.
Br J Cancer. 1999 Mar;79(7-8):1025-31. doi: 10.1038/sj.bjc.6690164.
Modulation of tumour cell growth by tumour-infiltrating leucocytes is of high importance for the biological behaviour of malignant neoplasms. In melanoma, tumour-associated macrophages (TAM) and tumour-infiltrating lymphocytes (TIL) are of particular interest as inhibitors or enhancers of cell growth. Recruitment of leucocytes from the peripheral blood into the tumour site is mediated predominantly by chemotaxins, particularly by the group of chemokines. The aim of this study was to identify peptides released by human melanoma cells with monocyte chemotactic properties. To assure the presence of biologically active mediators, biochemical purification and biological characterization of peptides was based on a detection system dependent on bioactive, monocyte chemotactic activity in vitro. Cell culture supernatants of melanoma cells were fractioned by heparin-sepharose followed by preparative reversed-phase HPLC steps to enrich monocyte chemotactic activity in one single band on a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel. These purified fractions were shown to react with RANTES-specific antibodies in an enzyme-linked immunosorbent assay (ELISA) as well as in Western blot analysis. Amino acid sequencing of the N-terminal protein fragment confirmed 100% homology to the RANTES protein. Further analysis showed that four out of eight melanoma cell lines constitutively expressed and secreted the beta-chemokine RANTES as detected by ELISA. The amount of RANTES protein secreted (up to 50 ng ml(-1)) was about 5-50 times higher than interleukin 8 (IL-8), determined in the same supernatant samples. Tumour necrosis factor alpha, (TNF-alpha), not, however, IL-2, interferon-gamma (IFN-gamma), or (alpha-melanocyte-stimulating hormone (alpha-MSH) was able to up-regulate RANTES and interleukin 8 secretion. Furthermore, higher levels of RANTES secretion in vitro were associated with increased tumour formation upon s.c. injection of six human melanoma cell lines in nude mice. Our data provide evidence that a subset of melanoma cells express mRNA and secrete RANTES protein which may be partly responsible for the recruitment of monocytes, T-cells and dendritic cells into the tumours. However, transplantation experiments in nude mice suggest that effects of RANTES may also benefit tumour progression. Further studies are needed to dissect the underlying mechanisms.
肿瘤浸润白细胞对肿瘤细胞生长的调节作用对于恶性肿瘤的生物学行为至关重要。在黑色素瘤中，肿瘤相关巨噬细胞（TAM）和肿瘤浸润淋巴细胞（TIL）作为细胞生长的抑制剂或增强剂尤其受到关注。外周血白细胞募集到肿瘤部位主要由趋化因子介导，特别是趋化因子家族。本研究的目的是鉴定人黑色素瘤细胞释放的具有单核细胞趋化特性的肽。为确保生物活性介质的存在，肽的生化纯化和生物学特性鉴定基于一种依赖体外生物活性单核细胞趋化活性的检测系统。黑色素瘤细胞的细胞培养上清液先用肝素琼脂糖进行分级分离，然后进行制备性反相高效液相色谱步骤，以在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶上的一条带中富集单核细胞趋化活性。这些纯化的级分在酶联免疫吸附测定（ELISA）以及蛋白质印迹分析中显示与RANTES特异性抗体发生反应。N端蛋白片段的氨基酸测序证实与RANTES蛋白100%同源。进一步分析表明，通过ELISA检测，8种黑色素瘤细胞系中有4种组成性表达并分泌β趋化因子RANTES。在相同的上清液样品中测定，分泌的RANTES蛋白量（高达50 ng ml(-1)）比白细胞介素8（IL-8）高约5-50倍。肿瘤坏死因子α（TNF-α），但白细胞介素2、干扰素-γ（IFN-γ）或α-黑素细胞刺激素（α-MSH）不能上调RANTES和白细胞介素8的分泌。此外，体外较高水平的RANTES分泌与在裸鼠皮下注射6种人黑色素瘤细胞系后肿瘤形成增加相关。我们的数据表明，一部分黑色素瘤细胞表达mRNA并分泌RANTES蛋白，这可能部分负责单核细胞、T细胞和树突状细胞募集到肿瘤中。然而，裸鼠移植实验表明RANTES的作用也可能有利于肿瘤进展。需要进一步研究来剖析其潜在机制。
