Peterson E P, Martinez G M, Martinez-Zaguilan R, Perona R, Gillies R J
Departments of Biochemistry, University of Arizona, College of Medicine, Tucson 85724.
J Cell Physiol. 1994 Jun;159(3):551-60. doi: 10.1002/jcp.1041590319.
The role of intracellular pH (pHin) in the regulation of cell growth in both normal and transformed cells is a topic of considerable controversy. In an effort to study this relationship NIH 3T3 cells were stably transfected with the gene for the yeast H(+)-ATPase, constitutively elevating their pHin. The resulting cell line, RN1a, has a transformed phenotype: The cells are serum independent for growth, clone in soft agar, and form tumors in nude mice. In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum-independent growth, using defined media to investigate the effects of specific growth factors on the transfected and parental NIH 3T3 cells. While both cell lines show similar growth increases in response to platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF), they respond differently to insulin, insulin-like growth factor-I (IGF-I) and PDGF-AA. RN1a cells exhibit increased growth at nanomolar concentrations of insulin but the parental cells had only a relatively minor response to insulin at 10 microM. Both cell lines showed some response to IGF-I in the nanomolar range but the response of RN1a cells was much larger. Differences in insulin and IGF-I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF-AA. RN1a cells are relatively insensitive to stimulation by PDGF-AA and express fewer PDGF alpha receptors as shown by Northern blots and receptor-binding studies. We propose a unifying hypothesis in which the H(+)-ATPase activates a downstream element in the PDGF-AA signal transduction pathway that complements insulin and IGF-I signals, while leading to downregulation of the PDGF alpha receptor.
细胞内pH值（pHin）在正常细胞和转化细胞生长调节中的作用是一个颇具争议的话题。为了研究这种关系，NIH 3T3细胞被稳定转染了酵母H(+)-ATP酶基因，从而持续提高其细胞内pH值。由此产生的细胞系RN1a具有转化表型：细胞生长不依赖血清，能在软琼脂中克隆，并在裸鼠体内形成肿瘤。在本研究中，我们进一步对该系统进行表征，以便了解用这种质子泵转染如何导致不依赖血清的生长，我们使用限定培养基来研究特定生长因子对转染的和未转染的NIH 3T3细胞的影响。虽然两种细胞系对血小板衍生生长因子（PDGF）-BB和表皮生长因子（EGF）的反应都表现出相似的生长增加，但它们对胰岛素、胰岛素样生长因子-I（IGF-I）和PDGF-AA的反应不同。RN1a细胞在纳摩尔浓度的胰岛素作用下生长增加，但亲本细胞在10微摩尔浓度时对胰岛素只有相对较小的反应。两种细胞系在纳摩尔范围内对IGF-I都有一些反应，但RN1a细胞的反应要大得多。仅胰岛素和IGF-I受体数量的差异无法解释这些结果。这两种细胞系对PDGF-AA的反应也不同。如Northern印迹和受体结合研究所示，RN1a细胞对PDGF-AA的刺激相对不敏感，且表达较少的PDGFα受体。我们提出一个统一的假说，即H(+)-ATP酶激活了PDGF-AA信号转导途径中的一个下游元件，该元件补充了胰岛素和IGF-I信号，同时导致PDGFα受体下调。
