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Cell Growth & Differentiation Vol. 13, 441-448, September 2002
© 2002 American Association for Cancer Research

Increased K-Ras Protein and Activity in Mouse and Human Lung Epithelial Cells at Confluence

Wafa Kammouni1, Gayatri Ramakrishna1,2, Gunamani Sithanandam, George T. Smith, Laura W. Fornwald, Akira Masuda, Takashi Takahashi and Lucy M. Anderson3

Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702 [W. K., G. R., G. T. S., L. M. A.]; SAIC-Frederick, Inc., Frederick, Maryland 21702 [G. S., L. W. F.]; and Division of Molecular Oncology, Aichi Cancer Center Research Institute, 464-8681 Nagoya, Japan [A. M., T. T.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Although K-ras is frequently mutated in lung adenocarcinomas, the normal function of K-ras p21 in lung is not known. In two mouse (E10 and C10) and one human (HPL1D) immortalized lung cell lines from peripheral epithelium, we have measured total K-ras p21 and active K-ras p21-GTP during cell proliferation and at growth arrest caused by confluence. In all three cell types, total K-ras p21 increased 2- to 4-fold at confluence, and active K-ras p21-GTP increased 10- to 200-fold. It was estimated that 0.03% of total K-ras p21 was in the active GTP-bound state at 50% confluence, compared with 1.4% at postconfluence. By contrast, stimulation of proliferation by serum-containing medium did not involve K-ras p21 activation, even though a rapid, marked activation of both Erk1/2 and Akt occurred. At confluence, large increases, up to 14-fold, were seen in Grb2/Sos1 complexes, which may activate K-ras p21. In sum, increased protein expression and activity of K-ras p21 are associated with growth arrest, not with proliferation, in mouse and human lung cell lines.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Ras p21s are highly conserved guanine nucleotide-binding proteins that couple cell surface receptors to intracellular signaling pathways (1 , 2) . For example, phosphorylation of receptor tyrosine kinases leads to activation of exchange factors such as Sos which in turn promote activation of ras p21 by exchange of GTP for GDP. Ras-p21-GTP activates downstream effectors, and is then returned to basal state by intrinsic GTPase activity, stimulated by the associated regulatory protein GAP.4 Mutations in the K-ras oncogene are found in a significant percentage of human lung adenocarcinomas (3 , 4) , and with even higher frequencies in cancers of the colon and pancreas. A classical paradigm was developed for the mechanism by which mutant ras protein contributes to cancer development, based largely on studies of H-ras p21 in fibroblasts. According to this paradigm, oncogenic mutations in the protein, especially in regions specified by codons 12, 13, and 61 of the gene, prevent GTPase-dependent down-regulation; therefore, the protein remains permanently active and continuously passes proliferation and survival signals through downstream pathways such as raf/MAPK(Erk) and PI3K/protein kinase B(Akt) (5 , 6) . Normal ras can also be transforming in fibroblasts if highly overexpressed (7) .

Recently, however, it has become apparent that this attractive paradigm is an oversimplification in many situations. Activated ras p21 can lead to proliferation, apoptosis, growth arrest or differentiation depending on the cell type, type of incoming signal, and/or the duration, intensity and type of downstream effector signaling pathway (8, 9, 10, 11, 12) . Furthermore the ras isoforms are not equivalent. Although H- and N-ras knockout mice are viable (13 , 14) , the K-ras knockouts are embryonically lethal (15) , and the ras isoforms show differential activity with regard to transformation, activation of raf and PI3K, and other properties (16, 17, 18) . The signaling functions specific to K-ras p21 have been little studied, and almost nothing is known about the functioning of K-ras p21 in the lung epithelial cells that are the origin of lung adenocarcinoma.

Recently, it was found for both the rat (19) and the mouse (20) that K-ras p21 showed increasing expression in the fetal lung as development progressed, with a large increase as growth slowed and differentiation ensued in the later fetus; maximum levels were seen in the adult lung. Expression of K-ras p21 was found to be specifically in the alveolar type II cells, whereas N-ras was localized in bronchiolar cells and H-ras in alveolar macrophages (19) .

These findings were not consistent with a role for K-ras p21 in the proliferation of lung type II cells; this protein seemed rather associated with growth arrest and differentiated function. We have now pursued this question further by study of peripheral lung epithelial cells in culture, and in addition to total K-ras p21, we have measured the amount of activated, GTP-bound K-ras p21. The results confirm that, in both mouse and human lung cells, K-ras expression and activity is highest in growth-arrested, confluent cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Ras Activation Assay.
This assay is based on the principle that only activated GTP-bound ras p21 binds to the RBD of raf-1. The method was tested with mouse lung adenocarcinoma E9 cells, because these possess only mutant K-ras, which would be expected to have high constitutive activity. Beads with attached vector-expressed RBD were mixed with extracts of E9 cells and recovered by centrifugation, and the ras p21 was eluted and quantified by Western immunoblot with a specific monoclonal antibody. As shown in Fig. 1ACitation , no K-ras-GTP was detected in a control consisting of beads and lysate but no raf-RBD. A control consisting of beads and raf-RBD but no lysate was also negative (not shown). These controls were repeated in four additional experiments (not shown) and were uniformly negative. The assay was linear up to 300 µg of starting lysate protein. This linearity assures quantitative reliability of the assay. Total K-ras p21 in the 80% confluent E9 cells was quantified by a parallel immunoprecipitation (Fig. 1A)Citation ; an average of 62% of this was recovered as K-ras-GTP. This was reproducible in a second experiment (not shown), in which the average scan values for K-ras-GTP and for total K-ras in 300 µg of lysate protein were 1497 ± 620 and 2474 ± 502, respectively, i.e., 61% as K-ras-GTP.



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Fig. 1. Assay for active K-ras p21-GTP. A, lysates of 80% confluent E9 cells with 50, 100, or 300 µg of protein were incubated overnight at 4°C with 70 µg of GST-RBD attached to GSH-beads or with beads only. After pelleting and washing of the beads, the proteins were eluted, separated by electrophoresis, transferred to membranes, and probed with anti-K-ras p21 monoclonal antibody. No K-ras p21 was recovered with the beads-only control (Lane 1). The relative scan values were 250 ± 21 for 50 µg of protein, 646 ± 87 for 100 µg, and 1396 ± 221 for 300 µg. Total K-ras p21 from lysate with 300 µg of protein was recovered by immunoprecipitation (Last two lanes) and gave average scan values of 2264 ± 861. B, total K-ras p21 in nontransformed and malignant lung cells. Lysates (300 µg of protein) from five subconfluent cultures each of malignant E9 and nontransformed E10 cells were probed for total K-ras p21. This migrated slightly faster and was about 2-fold greater in amount in the E9 cells (average scan values 494 ± 68 versus 218 ± 31). C, amounts of active K-ras p21-GTP were determined in the same cultures as shown in B. An approximate 30-fold-higher level was observed in the E9 cells (average scan values, 1789 ± 208 versus 61 ± 8).

 
Total and Activated Ras p21 in Nontransformed E10 Cells.
Amounts of total K-ras p21 in subconfluent cells, as indicated by immunoprecipitation followed by blotting, were 50% less in the immortalized but nontransformed E10 cells, compared with the malignant E9 cells (Fig. 1B)Citation . The mutant K-ras p21 in the E9 cells migrated slightly faster than the wild-type K-ras protein in E10 cells (Fig. 1B)Citation . The amount of K-ras p21 in the activated, GTP-bound form was about 30-fold greater in the E9 compared with the E10 cells (Fig. 1C)Citation , as expected from the fact that mutant K-ras p21 lacks GTPase activity.

Increase in Total K-ras p21 and Ras-GTP in Lung Cells at Confluence.
In E10 cells, the amounts of total K-ras p21 increased progressively, by about 5-fold, from ~50% confluence through 2 days postconfluence, designated 100%+ (Fig. 2A)Citation . In the E9 cells, in which protein expression was high, total K-ras p21 increased ~2-fold at confluence (Fig. 2A)Citation . Active ras-GTP in the E10 cells increased about 20-fold between ~50% confluence and 2 days postconfluence in this experiment (Fig. 2B)Citation . The amount of ras-GTP increased in E9 cells by about 7-fold between 50% and 100%+ confluence. Similar results were obtained whether a fixed number of cells were plated and harvested on the 3rd, 4th, 5th, and 7th days as illustrated in Fig. 2Citation , or whether various cell densities were seeded so that the cells were 50, 70, 100, or 100%+ confluent on the 3rd day (data not shown).



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Fig. 2. Total and K-ras p21-GTP in E10 and E9 cells during culture growth and confluence. Separate assay blots for E10 and E9 cells are shown. A, total K-ras p21 increased in E10 cells during culture growth, with relative scan values of 142, 379, 388, and 670 at ~50, 70, 100, and 100% 2 days postconfluence (Lanes 1–4). In E9 cells, total K-ras p21 was relatively high at lower confluencies; relative scan values were 784, 668, 1012, and 1467 (Lanes 5–8). B, active K-ras p21-GTP also increased markedly during the growth of the E10 cultures and was especially high at postconfluence (100%, Day 3); relative scan values were 47, 204, 352, and 951 (Lanes 1–4). An increase in K-ras p21-GTP also occurred with culture density in the E9 cells; relative scan values were 207, 489, 1314, and 1434 (Lanes 5–8).

 
To confirm and quantify the increase in K-ras activity as the cells growth-arrested at confluence, four separate cultures of E10 cells were harvested at 100%+ confluence. There was an average 206-fold increase in K-ras-GTP in 100%+ versus ~50% confluent cells (Fig. 3A)Citation . Although good agreement among the quadruplicate samples suggested that differences in protein content were not a likely concern, the starting lysates were assayed for the total amount of Erk1 and Erk2 proteins as a reference point (Fig. 3B)Citation . When normalized to the content of Erk1 and Erk 2 in the samples, the increase in K-ras-GTP between 50 and 100%+ cultures was 204-fold.



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Fig. 3. Quantification of increases in total and active K-ras p21 at postconfluent growth arrest. A, in quadruplicate samples, K-ras-GTP was compared at 50% versus 2 days postconfluent cultures (100%+). Average scan values were 2.1 ± 0.6 versus 429 ± 51, an average fold increase of 206.5. B, Erk1/2 protein levels were determined in the starting lysates used for A, above, to confirm equality. Average scan values for K-ras-GTP normalized to Erk1/2 protein were 1.1 ± 0.3 and 224 ± 26, an average fold increase of 203.6. C, for direct comparison of these samples, the following amounts of starting-lysate protein were loaded as indicated on the gels: 1 mg for K-ras-GTP, 50%; 500 µg for K-ras-GTP, 100%+; 40 µg for total K-ras p21, 50%; and 20 µg for total K-ras p21, 100%+. Average scan values were 4.2 ± 1.2, 411 ± 87, 573 ± 76, and 1171 ± 162. After correction for amounts of protein loaded, the relative amounts were 1:196:3411:13,940.

 
To evaluate directly the relative changes in total and active K-ras p21, for the samples presented in Figs. 3, A and BCitation , different amounts of protein from immunoprecipitates for total K-ras p21 and from the RBD fraction for K-ras-GTP were loaded onto the same gel, to obtain detectable signals with the same exposure time (Fig. 3C)Citation . After correction for the different amounts of protein, the amount of total K-ras p21 increased on average by a factor of 4.1 in postconfluent compared with 50% confluent cultures. This value is in good agreement with that obtained in the experiment illustrated in Fig. 2ACitation . The amount of K-ras-GTP increased on average by a factor of 196, in good agreement with the value from the assay illustrated in Fig. 3ACitation and higher than that from the previous experiment (Fig. 2B)Citation . Experiment-to-experiment variation in the relative increase in total and active K-ras p21 appeared to relate in part to the necessarily subjective decision as to when cultures were 50% confluent.

The concurrent quantification of total and active K-ras p21 (Fig. 3C)Citation permitted estimation of the percentage of the total that was in the active GTP-bound state. In the 50% confluent cells, 0.03% of the total K-ras p21 was in the GTP-bound state; this increased to 1.4% at postconfluence. Thus, both total K-ras p21 and the percentage of this in the active form increased with postconfluence growth arrest.

Increase in Total and Active K-ras p21 at Confluence in Other Mouse and Human Lung Cell Lines.
To test whether these differences would pertain in another immortalized mouse lung line and in an immortalized human lung cell line from the peripheral epithelium, the assays were repeated with C10 mouse and HPL1D human cells (Fig. 4)Citation . In C10 cells the increases between 50 and 100% confluency were about 3.8-fold for K-ras p21 and 9.3-fold for K-ras-GTP (Fig. 4, A and B)Citation , with no change in the average amount of total Erk1 + Erk 2 (Fig. 4C)Citation . Similarly, for human HPL1D cells, total K-ras p21 increased 3.7-fold and K-ras-GTP 8.7-fold, in 100% versus 50% confluent cells (Fig. 4, D and E)Citation , with no change in the average amount of {alpha} tubulin (Fig. 4F)Citation . These increases in total K-ras are similar to the comparable values for E10 cells at 50 versus 100% confluency (see Figs. 2Citation and 3Citation ). As for E10 cells, the relative increase in active K-ras-GTP with confluence of C10 and HPL cells was greater than the increase in total K-ras p21. Thus, an increase in both total K-ras and percentage of active K-ras, with growth arrest at confluency was a general characteristic of mouse and human lung peripheral epithelial cells.



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Fig. 4. Total and K-ras p21-GTP in mouse C10 and human HPL1D cells during culture growth and confluence. A and B, at left, the p21 standard. A, total K-ras p21 in C10 cells at 50 and 100% confluence; relative average densitometric scan values were 102 ± 30 and 389 ± 70, respectively. When normalized to total Erk1/2 protein (C), relative scan values were 64 ± 11 and 238 ± 21, respectively. B, activated K-ras p21-GTP in C10 cells; relative scan values were 14 ± 13 at 50% confluence and 130 ± 43 at 100% confluence. Normalized to total Erk1/2 protein (C), these were 8.5 ± 8.2 and 81 ± 20, respectively. C, the lysates used for the results shown in A and B were probed for Erk1/2 total protein, confirming approximately equal protein content. D, total K-ras p21 in HPL1D immortalized human lung cells at 50% confluency, relative scan value average 153 ± 46, and at 100% confluency, 564 ± 134. Normalized to {alpha}-tubulin protein (F), these values were 74 ± 23 and 279 ± 65, respectively. E, activated K-ras p21-GTP in HPL1D cells, relative scan values 68 ± 35 for 50% confluent and 596 ± 37 for 100% confluent. These values were 33 ± 7 and 283 ± 9 after normalization to {alpha}-tubulin (F). F, {alpha}-tubulin loading controls for the HPL1D cell extracts used for the data in D and E.

 
Serum Activation of Erk1/2 and Akt but not K-ras p21.
Subconfluent E10 cells were serum starved in 0.1% serum-containing medium for 48 h. After replacement with complete medium, which reinitiates the cell cycle, total K-ras p21 and K-ras-GTP were measured at intervals of 1.0 min to 12 h. Data through 6 h are shown in Fig. 5Citation . No additional changes were observed to 12 h (not shown). Neither total K-ras p21 (Fig. 5A)Citation nor K-ras-GTP (Fig. 5B)Citation increased after serum stimulation during this time. Amounts of total Erk1/2 in these lysates were similar at all of the time points, confirming the constancy of the protein loaded (Fig. 5C)Citation . The addition of serum led to rapid increase in amounts of active, phosphorylated Erk1/2, which reached a maximum at 10 min and remained at this level for 4 h (Fig. 5D)Citation . Thus, there was an increase in activated Erk1/2, after the serum stimulation of E10 cells, that was not associated with increased activation of K-ras 21.



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Fig. 5. Serum activation of Erk1/2 but not K-ras p21 in E10 cells. After growth arrest of 60% confluent E10 cells by serum-starvation for 48 h, complete medium with serum was restored. Lysates at each time point were used for assay of total K-ras p21 (A), K-ras-GTP (B), total Erk1/2 (C), and activated phosphorylated Erk1P/2P (D). B, at the left, the p21 standard. Serum stimulation did not lead to an increase in K-ras-GTP (B), whereas marked activation of Erk1/2 was observed (D). In a separate experiment, similar cultures were followed for 12 h and no change in total K-ras or K-ras-GTP was observed (not shown).

 
Serum activation of Erk1/2 was further examined in C10 and HPL cells and in confluent cultures. In addition, we assayed Akt, which is often activated by phosphorylation after serum stimulation and which has been implicated as a downstream target of ras p21 in some systems. The addition of serum-containing medium to serum-starved E10, C10, or HPL1D cells led to a rapid, marked increased in phosphorylated (activated) Erk1 and Erk2 (Fig. 6, A, B, and C)Citation and in phosphorylated Akt (Fig. 6, D, E, and F)Citation . Increases were observed within 1 min and appeared to be a maximum at 5 min. The exception was proliferating HPL1D cells, which appeared to have constitutive Akt activation even under serum-starved conditions. Total Erk1/2 and Akt protein did not change over this time interval, and neither total nor activated Erk1/2 or Akt were altered in amount in confluent, compared with subconfluent, cell cultures. This was true of unmanipulated cultures, as well as those after serum starvation (not shown). Thus, Erk1/2 and Akt were quite dissimilar to K-ras p21 with regard to expression and activation patterns.



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Fig. 6. Activation by serum of Erk1/2 and Akt in E10, C10, and HPL1D cells. Proliferating (50%) and confluent (100%) cells were serum starved for 48 h (AC). Total and activated Erk1/2 in E10 (A), C10 (B), and HPL (C) cells. Total Erk1/2 did not change with serum stimulation, and Erk1/2 were rapidly activated to Erk1P/2P in both proliferating and confluent E10, C10, and HPL cells. D-F. Total and activated phosphorylated pAkt in E10 (D), C10 (E), and HPL (F) cells. Total Akt did not change with serum stimulation, and pAkt rapidly increased in both proliferating and confluent E10, C10, and HPL cells after serum stimulation.

 
Formation of Grb2/Sos-1 Complex.
Because receptor-mediated activation of ras p21 is often accomplished by formation of a Grb2/Sos1 complex, we assessed levels of this complex as correlated with K-ras activation at confluence, in E10, C10, and HPL1D cells. As shown in Fig. 7Citation , in all three of the cell types, the amounts of Grb2 and Sos1 proteins showed only minor increases in the cells at confluence, up to 2-fold. In contrast, a large increase in the Grb2/Sos1 complex was observed in the E10 cells (14-fold; Fig. 7ACitation ) and the human HPL1D cells (5-fold; Fig. 7CCitation ). Results with the C10 cells were more variable and fell short of statistical significance (Fig. 7B)Citation .



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Fig. 7. Increase in Grb2/Sos1 complex at confluence. A, in E10 cells at 100% confluence, compared with 50% confluence, total Grb2 increased slightly (top panel), from 2409 ± 450 relative scan units to 3499 ± 667 (P = 0.08). Total Sos1 doubled, from 989 ± 379 average scan units at 50% to 1882 ± 199 at 100% (P < 0.05; second panel). Grb2/Sos1 association increased 14-fold, from 182 ± 53 to 2593 ± 91 (P < 0.01; third panel). B, in C10 cells, total Grb2 and Sos1 proteins did not change significantly with 100% confluence: 4974 ± 54 relative scan units for Grb2 at 100% versus 4061 ± 1434 for 50% (top panel) and 1480 ± 145 versus 1692 ± 1127 for Sos1 (middle panel). Grb2/Sos1 complex increased at confluence, 925 ± 219 relative scan units versus 708 ± 282 at 50% (lower panel), although this difference was not of statistical significance (P = 0.35). C, in human HPL1D cells at 100% confluence, compared with 50% confluence, total Grb2 increased slightly but significantly (1863 ± 179 to 2440 ± 91; P < 0.01; top panel). Sos1 total doubled at 100% confluence from 1430 ± 84 to 2813 ± 404 (P < 0.01; second panel). Grb2/Sos1 complex increased 6-fold at 100% confluence, from 396 ± 134 to 2284 ± 605 (P < 0.01; third panel).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The results of this study provided confirmation of in vivo findings, to the effect that K-ras expression and activation in lung peripheral epithelial cells are higher in growth-arrested cells than in those that are rapidly proliferating. The increase in K-ras activation at confluence was paralleled by an increase in Grb2-Sos1 complex, consistent with Grb2 participation in mediating the K-ras p21 activation in response to signals generated at the cell membrane. An increase in activated, phosphorylated Erks, or in phosphorylated Akt, was not observed at confluence, although rapid increase in both was easily demonstrated within a few minutes of serum treatment of serum-starved E10 cells. Serum did not activate K-ras p21.

These results pose many interesting questions for further study. Does activated K-ras p21-GTP lead to growth arrest, or is it a consequence of cell cycle exit with the purpose of upregulating differentiation programs? To what upstream signal(s) does K-ras respond? What pathway carries the signal to the nucleus, since activated Erks and Akt appear not to be involved?

Because overexpressed activated ras genes have repeatedly been shown to impose growth arrest or senescence when transfected into primary cells (9 , 11 , 21) , it is reasonable to propose a similar consequence of increase in K-ras p21 amount and activity in lung type II cells. Candidate upstream effectors, leading to an increase in amount and activity of K-ras p21, could include intercellular junctions, adhesion molecules such as integrins (22 , 23) , and/or paracrine/juxtacrine factors that increase in concentration as cell density increases.

The best growth factor candidates at present may be TGF-ß and IGFII. TGF-ß can rapidly activate ras p21 in cultured cells, including lung cells (24) . It inhibits growth of the HPL1 human lung cells used in our experiments (25) as well as growth of nontransformed mink lung epithelial cells (26) and some, although not all, transformed mouse lung cell lines (27) . A dominant negative type II TGF-ß1 receptor in mice was associated with enhanced lung tumorigenesis (28) . This receptor was found to be more expressed in E10 cells than in transformed mouse lung cell lines (27) . TGF-ß1 mRNA was lacking in exponential E10 cells (29) but has not been examined in confluent or growth-arrested cells.

IGFII is also a possible candidate. In rat lung type II cells in culture, growth arrest was associated with increased IGFII, type 2 IGF receptor, and IGF-binding protein 2 in the medium (30 , 31) . We have found high expression of IGFII mRNA in E10 cells, with a 3-fold increase at confluence.5 Although IGFII is a fetal growth factor and often is upregulated in neoplasms (32) , it also can act as a differentiation factor in several types of tissue (33) , and tumor suppressive effects have also been described (34) . IGFII is known to signal through the IGFI receptor, which, as a tyrosine kinase, has the potential to activate ras p21 (35) . However, this receptor has consistently been associated with tumor stimulation rather than with suppression (35) . The IGFII receptor, although generally regarded as down-regulating IGFII, has not been ruled out as participating in signal transduction (36) . This receptor has been implicated as a tumor suppressor (37) , and mutations in it were recently reported in squamous cell carcinomas of lung (38) . Furthermore, because the IGFII receptor activates latent TGF-ß1, synergism between these two growth factors may be possible. On the other hand, IGFII is a likely constituent of FCS, which did not activate K-ras p21 in our cells.

Other agonists for membrane tyrosine kinase receptors should also be considered, because the increase in Grb2/Sos1 complex at confluence suggests their involvement. Receptors activating Grb2 include EGF receptor, Ret, Met, ErbB2, and the insulin receptor (39 , 40) . The EGF receptor and PDGF receptor {alpha} increased in E10 and C10 cells at confluence and were absent from most transformed cell lines (41) . Increase in the malignant phenotype of mouse lung tumors after sialoadenectomy, which reduces circulating EGF (42) , could imply a tumor-suppressive role for EGF. PDGF receptor {alpha} is a growth-arrest-specific (gas) gene in fibroblasts (43) . However, both EGF and PDGF were growth-stimulatory for E10 cells (41) . Fibroblast growth factor 2 is also a possibility, because it stimulated surfactant production by fetal rat lung cells, using a MAPK-independent pathway (44) .

The pathway(s) possibly used by the increased K-ras p21 activity to bring about growth arrest and/or differentiation are unknown at present. Lack of any correlation with activation of the PI3K/Akt and raf/Erk1/2 pathways makes these unlikely downstream targets. Other possibilities include GAP, ralGDS, MEKK1/jun kinase, protein kinase C{zeta} and others, with potential for effects on gene expression and/or cytoskeletal organization (10 , 11 , 45) . No studies have been reported of these signaling components related specifically to K-ras p21 in lung.

How may cell cycle arrest putatively be effected by K-ras activation? High expression of transfected oncogenic or wild type K-ras in various cells, with or without growth arrest or senescence, has been associated with up-regulation of p16, p21waf1/cip1, p53, and Gadd45 (45, 46, 47, 48) . pRb increased during fetal lung cell growth arrest and lung maturation (49) , and this Rb mRNA had reduced expression in mouse lung tumors (50 , 51) . p16 abnormalities are common in these tumors (52, 53, 54) . Growth arrest induced in lung alveolar epithelial cells by glucocorticoids was associated with up-regulation of p21Waf1 (54) . The cyclin-dependent kinase inhibitor p27Kip1 seems particularly likely to have a central role. More lung tumors developed in p27 nullizygous and heterozygous knockout mice (55) . There was a specific increase in p27 in malignant mouse lung E9 cells, the transformed phenotype of which was partially reversed by transfection with connexin (56) . A high percentage of human non-small cell lung carcinomas showed reduced levels of p27, and this correlated with poor prognosis (57, 58, 59) . In our cultured E10 cells, p27 was markedly up-regulated at confluence (not shown). Interestingly, G1 arrest in several malignant cell lines was associated with p27 up-regulation independent of MAPK (60) , reminiscent of the lack of Erk1/2 activation at confluence in our cells. On the other hand, p27 was down-regulated during G1 growth arrest by glucocorticoids in rat lung cells (54) , and, in mink lung cells, suppression of the cell cycle with TGF-ß did not involve an alteration in total p27, although its complexes were altered (61) .

If activated K-ras p21 is related to growth arrest and differentiation, how do mutations in it, leading to continuous activation, contribute to tumorigenesis? One possibility is that mutant, oncogenic K-ras p21 interacts abnormally with downstream pathways not utilized by normal K-ras p21, such as jun and jun kinase (62) . Another is that the mutation prevents interaction with a downstream effector that leads to growth arrest/differentiation, e.g., GAP.

In summary, the control of the increase in total and active K-ras p21 during growth arrest in lung type II cells, and the consequences of this increase in the maintenance of growth arrest and differentiation, are important topics for further research, to reveal critical targets for intervention in lung cancer initiation, development, and malignant progression.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines and Culture Conditions.
Mouse lung type II cell lines, immortalized E10 and C10 and malignant E9, originally derived from cultured primary BALB/c mouse lung cells (63) , were obtained from Dr. Randall Ruch, Medical College of Ohio, Dr. Sonia Jakowlew, Medicine Branch, National Cancer Institute, and Dr. Alvin Malkinson, University of Colorado, respectively. E9 and E10 cells were maintained in Connaught Medical Research Laboratories (CMRL) 1065 basal medium and C10 cells in DMEM medium (BioFluids Co., Rockville, MD) supplemented with 2 mM glutamine, penicillin/streptomycin, and 10% FCS (Gemini Bio-products Co., Calabasas, CA; selected for the ability to stimulate vigorous growth in these cells). HPL1D immortalized human lung cells from the peripheral epithelium, with characteristics of type II and Clara cells, were maintained as described previously (25) in Ham’s F-12 medium buffered with 15 mM HEPES (pH 7.3; BioFluids) and supplemented with 5 µg/ml bovine insulin (Sigma Chemical Co., St. Louis, MO), 5 µg/ml human transferrin (Cal Biochem Co., La Jolla, CA), 10-7 M hydrocortisone (Sigma), 2 x 10-10 M triiodine thryonine (Sigma), 100 IU/ml penicillin (BioFluids), 100 µg/ml streptomycin (BioFluids), 25 mg/ml fungizone (BioFluids), and 3% FCS (Gemini BioProducts). Stages of cell culture were estimated visually. Cells on the day of confluence were designated 100% confluent. Postconfluent cells were designated Day 3 or 100%+, and were collected 2 days later, with a change of medium on the intervening day. Cells were removed from the plates by scraping.

Immunoblot Assays.
Lung cells were scraped in lysis buffer containing 25 mM HEPES, 1 mM EDTA, 1 mM MgCl2, 1% NP40, 0.25% sodium deoxycholate, and the protease inhibitors phenylmethylsulonyl fluoride, 1 mM, and aprotinin, 0.2 trypsin inhibitory units (64) . The extracts were loaded at the protein concentrations indicated in the figure legends on Tris-glycine gels (Novex, Inc., San Diego, CA) at 12% for K-ras p21 and Grb2, 8% for Erk1/2 and Akt, and 6% for Sos-1, and were electrophoresed at 100 V. After transfer to Hybond nitrocellulose membranes (Amersham, Buckinghamshire, Unitied Kingdom) at 22 V at room temperature, the blots were blocked with 2% bovine serum albumin for 2 h at room temperature and probed overnight with monoclonal antibodies at 1 µg/ml anti-K-ras (Oncogene Science, Inc., Uniondale, NY) or anti-Grb-2 or anti-Sos-1 (Transduction Laboratories, San Diego, CA). Total and phospho Erk1/2 and total and phospho Akt were detected with a PhosphoPlus p44/p42 MAP Kinase (Thr202/Tyr204) antibody kit and Akt Kinase assay kit from New England Biolabs (Beverly, MA). The blots were incubated for 60 min at room temperature with secondary antibody (goat antimouse IgG conjugated with horseradish peroxidase in 1% gelatin, 1:2000; Amersham Co., Amersham, United Kingdom). After repeated washings in buffer containing 0.5% Tween 20, the blots were developed using a chemiluminescence ECL kit (Amersham) and exposed to X-ray film. Assessment of the equality of amounts of protein in the starting lysates used total Erk1 + 2 protein for E10 and C10 cells and {alpha}-tubulin for HPL1D cells. {alpha}-Tubulin, a more conventional loading control, was found to show some variability with cell culture stage in E10 and C10 cells.

Immunoprecipitation of K-ras p21.
Cells were prepared in lysis buffer, as described above, and lysate containing 250 µg of protein were immunoprecipitated with 30 µg of anti-v-Ha-ras-agarose-conjugated antibody (Oncogene Science) for 12 h at 4°C. This reagent was used because no antibodies specific to K-ras p21 are effective for immunoprecipitation. This amount of agarose was determined to be adequate for immunoprecipitation of all of the K-ras 21, as described previously (64) . The immunoprecipitate was washed three times in buffer prior to electrophoresis and immunoblotting as described above.

K-ras-GTP Assay.
This assay involved the use of the RBD of raf-1 fused to glutathione S-transferase (GST) as described previously (65 , 66) . The expression vector encoding the fusion protein GST-RBD was kindly provided by Dr. Stephen Taylor (Cornell University, Ithaca, NY). Total cell lysate (300 µg of protein) was incubated with 50–70 µg of GST-RBD bound to GSH-beads overnight at 4°C. The beads were pelleted by centrifugation and washed three times with lysis buffer. The proteins were eluted by boiling in 2x Laemmli’s buffer (67) containing 0.5 M DTT and subjected to electrophoresis and immunoblotting as described above. Controls consisting of GSH-beads and lysate but no GST-RBD, or GSH-beads and GST-RBD but no lysate, were included during methods development and periodically during experimentation and were consistently negative (see Fig. 1Citation ).

Assay for Grb-2/Sos-1 Association.
Cell lysate (500 µg of protein) was immunoprecipitated with anti Grb-2 polyclonal antibody (1 µg/ml; Santa Cruz Laboratories, Santa Cruz, CA) overnight at 4°C. The immunocomplex was captured using protein A-agarose beads (Amersham) by shaking at 4°C for 2 h. After three washes with lysis buffer, the immunocomplex was eluted from the beads by boiling in Laemmli’s buffer for 10 min. The proteins were separated on 6% Tris-glycine gels and blotted to membranes as described above, and were probed with anti-Sos-1 monoclonal antibody (1 µg/ml; Transduction Laboratories).

Quantification and Statistical Analysis.
Bands on X-ray films were quantified with an ImageQuant densitometric scanner. Values are presented as relative scan units for each blot. Parametric or nonparametric statistical analyses were carried out as appropriate, using version 3.0 Instat software from GraphPad Software, Inc., San Diego, CA.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 W. K. and G. R. contributed equally. Back

2 Present address: Centre for DNA Fingerprinting and Diagnostics, ECI1 Road, Hyderabad-500 076 AP, India. Back

3 To whom requests for reprints should be addressed, at National Cancer Institute at Frederick, Building 538, Room 205B, Fort Detrick, Frederick, MD 21702. Phone: (301) 846-5600; Fax: (301) 846-5946; E-mail: Andersol{at}mail.ncifcrf.gov Back

4 The abbreviations used are: GAP, GTPase-activating protein; RBD, ras binding domain; TGF, transforming growth factor; IGFII, insulin-like growth factor II; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; GST, glutathione s-transferase; GSH, reduced glutathione. Back

5 W. Yu, unpublished observation. Back

Received for publication 11/30/00. Revision received 5/14/02. Accepted for publication 7/15/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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