Our previous studies8–10 demonstrated that the forced expression of oncogenic KRAS induced severe growth suppression in primary human airway epithelial cells, and also in Simian virus 40 large T antigen (SV40LT)—immortalized human airway epithelial cell lines.8–10 Similarly, others reported that the forced expression of oncogenic HRAS and/or KRAS markedly suppressed the growth of primary human fibroblastic cells61–63 and/or some human neuroblastoma cell lines,64,65 supporting that RAS mutations alone are insufficient to transform human cells and also suggesting that RAS could function as a tumor suppressor in certain instances.
Two types of oncogenic RAS-induced growth suppression and/or cell death, namely premature senescence66–69 and autophagy (type II programmed cell death),70,71 have been recognized. In addition, apoptosis induced by certain stimuli such as treatment of chemotherapeutic agents,72,73 cytokines,66,74–76 and depletion of growth factors,66 is also promoted through the RAS-mediated signaling pathways. Premature senescence is a complete growth arrest characterized by a marked flattening and enlargement of cells.61–63,77 Autophagy is cell death with cytoplasmic vacuolization.66,70,71 Apoptosis is cell death with nuclear shrinkage and fragmentation.66,78 The three could be mediated through different, but partly common, signaling pathways.66,77 Transcriptional activation of p16INK4A by ETS2 through the RAS-MAPK pathway was found to be essential to oncogenic RAS-induced premature senescence in primary fibroblastic cells.61–63,69,77,79,80 p53's activation by ataxia telangiectasia gene product (ATM) through the RAS-MAPK pathway was also reported to be essential to the induction of premature senescence, autophagy and apoptosis.62,63,66,75–77,81–83 Moreover, disruption of the machinery for controlling the cell cycle by oncogenic RAS, caused DNA-damage that triggered activation of the p53 pathway, resulting in apoptosis.77,84 On the other hand, the RAS-PI3K-mediated signal cascade was found to be crucial to a specific type of cell death similar to autophagy in some neuroblastoma cell lines.64,65 As described above, oncogenic KRAS induced severe growth suppression in primary and also SV40LT-immortalized human airway epithelial cells.9 The cells were markedly enlarged and had many vacuoles of various sizes in their cytoplasm (Fig. 1a).9 This morphologic change was consistent with autophagy.8 The SV40LT protein is known to inactivate both p53 and retinoblastoma protein (RB).85 Oncogenic KRAS induced autophagy-like growth suppression also in H1299 lung cancer cells whose p53 was inactivated through homozygous deletion and p16INK4A gene was inactivated through the promoter's hypermethylation.9,86 Also, oncogenic KRAS induced the same type of growth suppression in some other lung cancer cell lines whose p53 and p16INK4A was inactivated.9 KRAS mutations have been found in 25% to 35% of AAHs, but the inactivation of both p53 and p16INK4A merely accompanies.1,3,6,11,57 Moreover, the forced expression of a constitutively active phosphoinositide 3-kinase catalytic α (PIK3CA), a catalytic subunit of PI3K, did not induce growth suppression in immortalized airway cells and some lung cancer cells.87 Thus, p53, p16INK4A/RB, or PI3K is not indispensable to the oncogenic KRAS-induced growth suppression, and other unidentified effectors could be involved.
Figure 1. (a) Morphological change on oncogenic KRAS-induced growth suppression in a Simian virus 40 large T antigen-immortalized human airway epithelial cell line.9,95 Empty vector (MOCK), wild-type KRAS (G12), or mutated oncogenic KRAS (V12) was transduced. After an appropriate period of selection during which cells not gene-transduced died, surviving cells were fixed and stained with the Papanicolaou method. The cells transduced with oncogenic KRAS (V12) were markedly enlarged and flattened, and had vacuoles in their cytoplasm. Magnification is ×400, each. (b) The selected cells were grown and passed several times. The means and standard deviations (error bars) of cumulated population doublings (PDLs) from triplicate experiments are presented.
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