مرکزی صفحہ Lung Cancer K-ras 4A and 4B mRNA levels correlate with superoxide in lung adenocarcinoma cells, while at the...

K-ras 4A and 4B mRNA levels correlate with superoxide in lung adenocarcinoma cells, while at the protein level, only mutant K-ras 4A protein correlates with superoxide

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جلد:
80
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english
رسالہ:
Lung Cancer
DOI:
10.1016/j.lungcan.2013.01.022
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June, 2013
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Lung Cancer 80 (2013) 263–269

Contents lists available at SciVerse ScienceDirect

Lung Cancer
journal homepage: www.elsevier.com/locate/lungcan

K-ras 4A and 4B mRNA levels correlate with superoxide in lung adenocarcinoma
cells, while at the protein level, only mutant K-ras 4A protein correlates with
superoxide
Richard J. Calvert a,∗ , Meghana Gupta b , Anna Maciag c , Yih-Horng Shiao b , Lucy M. Anderson b
a

U.S. Food and Drug Administration, MOD-1 Laboratory, 8301 Muirkirk Road, Laurel, MD 20708, United States
Laboratory of Comparative Carcinogenesis, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States
c
Basic Science Program, SAIC-Frederick Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, United States
b

a r t i c l e

i n f o

Article history:
Received 29 October 2012
Received in revised form 18 January 2013
Accepted 26 January 2013
Keywords:
K-ras 4A
Lung
Adenocarcinoma
Superoxide
Mutant K-ras
Cell lines
siRNA

a b s t r a c t
The K-ras gene is frequently mutated in lung and other cancers. K-ras protein includes two splice variants,
K-ras 4A and 4B. While K-ras 4B is more widely expressed, recent evidence implicates K-ras 4A in lung
tumorigenesis. We found that K-ras 4A protein has a wide range of expression in a large panel of human
lung adenocarcinoma cell lines. In cell lines with mutant K-ras, but not those with wildtype K-ras, the
K-ras 4A protein had a strong positive correlation with levels of cellular superoxide. We investigated
whether K-ras 4A protein was involved in superoxide production, or alternatively was modulated by
elevated superoxide. Experiments with small interfering RNA targeting K-ras 4A did not confirm its role
in superoxide generation. However, decreasing cellular superoxide with the scavenger Tiron tended to
reduce levels of K-ras 4A protein. K-ras 4A and 4B mRNA were also quantified in a number of NSCLC cell
lines. 4A mRNA correlated with 4A protein only in K-ras-mutant cells. K-ras 4A mRNA also correlated
with s; uperoxide, but with no difference between cell lines with mutant or wildtype K-ras. K-ras 4B mRNA
correlated with 4A mRNA and with superoxide, in both K-ras mutant and wildtype cells. The results are
consistent with superoxide directly or indirectly up-regulating expression of all K-ras genes, and also
increasing the stability of K-ras 4A mutant protein selectively.
Published by Elsevier Ireland Ltd.

1. Introduction
K-ras protein has two distinct isoforms, K-ras 4A and 4B, which
result from alternate splicing and differ mainly at the carboxyl
terminal hypervariable region; mutations at codons 12 and 13,
common in some human cancers, affect both isoforms. K-ras 4B
is more widely expressed [1,2]; is necessary for embryonic development, whereas K-ras 4A is not [3]; and does not affect lifespan
or spontaneous tumor incidence in mice [4]. In some situations, Kras 4A was found to be pro-apoptotic [4] or tumor suppressive [5].
For these reasons K-ras 4B has often been the main focus of attention. Recently, however, in mouse lung K-ras 4A was implicated in
tumor initiation [6] and development [7]. Increased K-ras 4A/4B
ratios in mouse lung correlated with susceptibility to lung tumorigenesis [1]. Cell culture studies also indicated enhanced oncogenic
properties for K-ras 4A: transfected into NIH3T3 fibroblasts, Rat1 fibroblasts, or RIE-1 epithelial cells, K-ras 4A was much more

∗ Corresponding author. Tel.: +1 301 210 6246; fax: +1 301 210 4699.
E-mail address: richard.calvert@fda.hhs.gov (R.J. Calvert).
0169-5002/$ – see front matter. Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.lungcan.2013.01.022

efficient in inducing transformed foci than 4B [8]. K-ras 4A, but not
4B, enabled anchorage independent growth of RIE-1.
Since K-ras is often mutated in lung adenocarcinomas, the
expression of K-ras 4A in these cancers is of interest. We have quantified expression of K-ras 4A protein and mRNA in a panel of human
lung adenocarcinoma cell lines with either wildtype or mutant Kras. We also examined the relationships between K-ras 4A protein,
mRNA, and superoxide levels in these cells, since a previous study
demonstrated a positive relationship between total K-ras protein
and superoxide [9]. The results suggest that superoxide may regulate levels of mutant K-ras 4A protein as well as all species of K-ras
mRNA.
2. Methods
2.1. Cell culture
Lung adenocarcinoma cell lines, obtained from ATCC (Manassas, VA), included H23, H441, H460, A549, H1355, H1373, H1734,
H1792, H1944, H2030, and H2122 with mutant K-ras and H322,
H1395, H1703, and H2126 with wildtype K-ras. A table of the characteristics of these cell lines may be found in [9]. Immortalized

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nontransformed HPL cells were obtained directly from Drs. A.
Masuda and T. Takahashi. These are nonmalignant and derived from
normal human peripheral lung epithelium, having characteristics
of both alveolar type II and Clara cells, and grown as described in
[10]. All other cells were grown in RPMI 1640 growth media (Quality Biological, Gaithersburg, MD) plus fetal bovine serum (10%, v/v),
364 ␮M glutamine, penicillin (90.9 units/ml), and streptomycin
(90.9 ␮g/ml). Cells were grown at 37 ◦ C in a humidified cell culture
incubator (7% CO2 concentration). Proliferating cells were used, at
70–80% confluence. Cells were scraped into lysis buffer containing
25 mM HEPES, pH 7.5, 1 mM EDTA, 10 mM MgCl2 , 1% NP-40 (nonyl
phenoxypolyethoxylethanol, USB, Cleveland, OH), 0.25% sodium
deoxycholate, 150 mM NaCl, 10% glycerol (Invitrogen, Carlsbad, CA)
1 mM phenylmethylsulfonyl fluoride, 0.2 TIU/ml aprotinin, 10 mM
NaF, and 0.2 mM sodium orthovanadate. Unless noted otherwise
above, chemicals were obtained from Sigma Chemical Company
(St. Louis, MO).
2.2. siRNA treatment
Cells were seeded at 4–6 × 105 cells/well in 6 well plates containing 2.3 ml of media. K-ras 4A was silenced using a proprietary
mixture of several siRNAs obtained from Santa Cruz Biotechnology
(Santa Cruz, CA), product number sc-43874. A 5 nM concentration of siRNA was found to produce the maximum degree of K-ras
4A silencing. The transfection reagent (HiPerFect) and nonsilencing siRNA (AllStars negative control siRNA) were obtained from
Qiagen (Germantown, MD). Cells were treated with siRNA for
72 h.
2.3. Superoxide measurement and scavenging
Methods for determination of superoxide using nitroblue tetrazolium (NBT) reduction were reported previously [9]. Intracellular
superoxide concentrations were reduced by incubating the cells in
media with 2–5 mM Tiron (4,5-dihydroxy-1,3-benzene-disulfonic
acid), Sigma–Aldrich, St. Louis, MO. Cells were treated with Tiron
for 72 h prior to collection.

antibody and ECl signal was detected as above. For determination
of comparative K-ras 4A protein values from different cell lines by
Western blotting, individual densitometric values were compared
to the mean densitometric value of two H441 standards on each
blot.
2.5. Quantitative real time RT PCR
Total RNA was collected using an RNeasy Mini Kit (Qiagen). For
K-ras 4A and 4B mRNA analysis, complementary DNA (cDNA) was
synthesized using the Omniscript Reverse Transcriptase Kit (Qiagen). SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) was used
for real-time PCR applications. The RT primer for K-ras 4A mRNA
had sequence 5 -ATCATCAACACCCAGATTAC-3 (nucleotides +768
to +749 relative to transcription start site). For the K-ras 4B, the
primer sequence was 5 -CATCATCAACACCCTGTC-3 (nucleotides
+645 to +628). PCR primers for amplification of either K-ras 4A
or 4B cDNAs had sequences 5 -GAGGCCTGCTGAAAATGACTG-3
(upstream, +169 to +189) and 5 -TTCGTCCACAAAATGATTCTGA3 (+275 to +254, downstream). GAPDH was used as an
internal standard. The GAPDH RT primer had sequence 5 GCAGGGGAGATTCAGTGTG-3 . PCR primers to amplify GAPDH
cDNA were 5 -GTGAAGGTCGGAGTCAACGGAT-3 (upstream) and
5 -CATGGGTGGAATCATATTGGAACA-3 (downstream). Quantitative analysis was performed using the Opticon Monitor 3.1 software
(Bio-Rad). Relative intensity of K-ras to GAPDH was calculated using
the delta Ct value, to facilitate comparison between cell lines.
2.6. Statistical analysis
Statistical analysis utilized GraphPad InStat 3 (www.
graphpad.com). Results were considered statistically significant at P < 0.05. Comparisons among multiple averages utilized the
Kruskal–Wallis non-parametric ANOVA followed by Dunn’s multiple comparisons test for pairwise comparisons. Other statistical
comparisons were made as described in the figure legends. For
correlations, the Pearson correlation values were calculated.
3. Results

2.4. Protein immunoblotting
Either Tris–glycine or bis–tris NuPage 12% polyacrylamide gels
(Invitrogen) were used to separate proteins. Gels were run with
both BenchMark and MagicMark XP protein standards (Invitrogen,
Carlsbad, CA). Running buffer was either 25 mM Tris–base, 192 mM
glycine with 0.1% SDS for Tris–glycine gels or MES NuPage buffer
(Invitrogen) for Nupage bis–tris gels. Voltage was 120 V for 1.5–2 h.
Gels were electro blotted on to a PVDF membrane for 1.5–2 h at 30 V
using Towbin’s transfer buffer (12 mM Tris–base, 96 mM glycine
and 20% methanol) or NuPage transfer buffer with 20% methanol
added.
Blots were blocked with 5% dry milk in PBS-T (phosphate
buffered saline with 0.1% Tween 20) for 1.5 h. After blocking,
blots were rinsed and incubated with a specific K-ras 4A antibody
(product # sc-522, Santa Cruz Biotechnology), diluted 1:200 in 3%
milk/PBS-T overnight at 4 ◦ C. Following 6 rinses with PBS-T over
60 min, the blot was incubated with an anti-rabbit secondary antibody coupled to horseradish peroxidase (GE Healthcare UK Ltd.,
Buckinghamshire, UK) diluted 1:1000 for 1 h at room temperature.
After rinsing as above, the blot was treated with ECL reagent (GE
Healthcare UK Ltd., Buckinghamshire, UK) and exposed to X-ray
film for 10 s to 10 min.
After three 1–2 min rinses with PBS-T, blots were incubated
with a ␤-actin antibody (# ab8227-50, Abcam, Cambridge, MA)
diluted 1:2.5 × 105 for 2 h at room temperature. Following three
15 min rinses in PBS-T, the blot was treated with a rabbit secondary

3.1. K-ras 4A protein correlated with superoxide in cell lines with
mutant K-ras
Two determinations of K-ras 4A protein from separate cultures
of cells grown at different times were made by Western blotting (Fig. 1). There were apparent major differences in amounts
of K-ras 4A protein among the cell lines. On average, levels were
not related to K-ras mutational status, with values of 0.17 ± 0.10
for wildtype (N = 5) and 0.18 ± 0.09 for mutant K-ras cell lines
(N = 11).
The averages of the two determinations of K-ras levels were
plotted vs. average superoxide values, and significance determined
by the Pearson linear correlation test. For the 11 cell lines presenting mutant K-ras, K-ras 4A protein correlated with average
superoxide levels with a high degree of significance (P = 0.0006,
r = 0.86) (Fig. 2A). By contrast, for the 5 cell lines with wildtype Kras, there was no significant correlation between K-ras 4A protein
and average superoxide levels (P = 0.85, r = 0.11) (Fig. 2B).
3.2. Inhibition of K-ras 4A protein levels by siRNA did not
decrease superoxide levels
We hypothesized that K-ras 4A protein might control or regulate superoxide generation. This possibility was tested in H441and
H1734 cells, both of which produced high levels of both K-ras
4A protein and superoxide. In H441 cells, 5 nM K-ras 4A siRNA

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265

Fig. 1. Levels of K-ras 4A protein in lung cancer cell lines, relative to that in H441 cells measured on the same immunoblot. A representative blot is shown in the inset. Each
value is the average of determinations with two different cell preparations, with the ranges given as bars.

decreased K-ras 4A to 35% of control value, P = 0.0001 (Fig. 3A).
Superoxide levels were not altered from control. Mean control
superoxide values of H441 cells vs. those treated with K-ras 4A
siRNA were 29.08 ± 2.15 and 30.28 ± 3.58 (mean ± SEM) nmol NBT
reduced/mg protein, respectively (Fig. 3B). These data were confirmed by a second siRNA experiment with H441 cells which
yielded similar results. For further confirmation of results, we did
the same experiment with H1734 cells. Five nM siRNA decreased
K-ras 4A protein expression to a mean of 61.4% of control value
(P = 0.0298, t-test). There was no significant difference in superoxide levels in control vs. siRNA-treated cells: 18.35 ± 2.039 and
17.93 ± 4.318 nmol NBT reduced/mg protein, respectively (data not
shown). Therefore, we concluded K-ras 4A was not responsible for
superoxide generation, at least in these two cell lines.
3.3. Decrease in superoxide levels tended to reduce K-ras 4A
protein levels

Fig. 2. Correlations of average relative K-ras 4A protein levels with average superoxide levels (␮mol nitro blue tetrazolium reduced in 30 min/mg total protein, from
[9]). Significance values from Pearson correlations are given on the graphs. Correlation was strong for cell lines with mutant K-ras (A), but lacking in those with
wildtype K-ras (B).

An alternative hypothesis was that cellular superoxide positively regulates K-ras 4A protein levels. Levels of superoxide were
reduced by incubation of H441 or H1734 cells with the superoxide
scavenger Tiron. In H441 cells, 5 mM Tiron significantly decreased
superoxide production by 26% (P = 0.0291, 2-tailed t-test, N = 9
for control, N = 8 for Tiron) and K-ras 4A protein expression by
18% (Fig. 4A and B). Because the decrease in K-ras 4A protein in
H441 cells was modest, Tiron K-ras 4A protein values on each gel
were normalized to the mean of the control values for that gel
(Fig. 4C) and statistical analysis utilized a 2-tailed 1 sample t-test
(P = 0.0476, n = 6 for both controls and Tiron-treated cells). H1734
cells were treated with 2 mM Tiron, to avoid the cytotoxicity we
noted at 5 mM. This tended to decrease superoxide production by
20%, which was not statistically significant (not shown, P = 0.126,
N = 5 for control, 5 for Tiron-treated). Despite the fact that this 20%

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decrease in superoxide was not statistically significant, a significant
40% decrease in K-ras 4A protein was noted with Tiron treatment
(not shown, P = 0.0406, N = 5 for control and 5 for Tiron-treated).
These results together suggested that superoxide was stimulating
up-regulation of K-ras 4A protein in these cell lines.
3.4. Levels of K-ras 4A protein and mRNA correlated in cell lines
with mutant K-ras

Fig. 3. (A) Representative Western blot for K-ras 4A following siRNA treatment in
H441 cells, resulting in a significant decrease of K-ras 4A levels to 35% of control
(P = 0.0001). Abbreviations: NT, no treatment; R, transfection reagent only; NS, nonsilencing siRNA (negative control); and si, K-ras 4A siRNA. (B) The reduction of K-ras
4A protein produced no change in superoxide. Units for superoxide are the same as
in Fig. 2.

To investigate possible mechanisms of effects of superoxide on
K-ras 4A protein, we quantified K-ras 4A mRNA in 6 cell lines,
including ones with high or low superoxide and mutant or wildtype
K-ras. These lines were characterized by variable levels of K-ras 4A
mRNA normalized to GAPDH mRNA (Fig. 5A). K-ras 4A mRNA levels were significantly higher in H441 cancer cells, compared with
the non-transformed HPL cells. Levels were also higher in H2126
and A549 cells by pair-wise tests. For those with mutant K-ras,
4A protein and 4A mRNA correlated strongly (Fig. 5B, P = 0.0001),
whereas those with wildtype K-ras showed no significant correlation (Fig. 5C, P = 0.53). These results suggest that the level of K-ras
4A mRNA is a limiting factor for amounts of K-ras 4A protein, specifically in cells with mutant K-ras.
3.5. K-ras 4A mRNA correlated with superoxide in all cell lines
The results above raised the possibility that superoxide might
be selectively regulating transcription of the mutant K-ras gene.
Superoxide correlated strongly with K-ras 4A mRNA (Fig. 5D,
P < 0.0001 for all 6 lines). But this was true for the lines with
wildtype K-ras (P < 0.0001) as well as for those with mutant K-ras
(P = 0.0013). These results suggest that superoxide might indeed
influence K-ras 4A mRNA levels, but do not explain why only
mutant K-ras 4A protein correlates with superoxide.
3.6. K-ras 4B mRNA levels correlated with K-ras 4A mRNA levels
and with superoxide

Fig. 4. (A) Representative Western blot of K-ras 4A protein from H441 cells treated
with 5 mM Tiron (C, control and T, Tiron). (B) Average superoxide values for these
cells (units defined in Fig. 2), *P = 0.0291. (C) Levels of K-ras 4A protein normalized
to the mean of controls for each blot, **P = 0.0476, two-tailed one sample t-test.

K-ras 4B mRNA levels were also measured in the cell lines
(Fig. 6). As for K-ras 4A, H441 cancer cells presented a significant increase in K-ras 4B mRNA relative to nontransformed HPL
cells. A549 cells had higher K-ras 4B mRNA, and H1944 lower,
by pairwise tests. Relative levels of K-ras 4A and 4B mRNAs were
correlated for all cell lines (P < 0.0001), for K-ras mutant cell lines
(P < 0.0001) (Suppl. Fig. 1A and C) and for K-ras wildtype cell lines
(P = 0.061 and 0.0017) (Suppl. Fig. 1B and D). It thus appeared
that 4A and 4B mRNAs were similarly regulated, whether or not
mutated.
As for K-ras 4A mRNA, K-ras 4B mRNA correlated significantly
with superoxide for all cell lines, with P = 0.017 for mutant cell lines
(Suppl. Fig. 2A), and with somewhat less significance (P = 0.044) for
wildtype cell lines (Suppl. Fig. 2B). Collectively these results are
consistent with superoxide regulating pre-splicing expression of
K-ras transcription.
Supplementary material related to this article found, in the
online version, at http://dx.doi.org/10.1016/j.lungcan.2013.01.022.
Levels of K-ras 4A and 4B mRNA were similar on average for cell
lines with wildtype or mutant K-ras, but 4A predominated in H441
cells.
Average levels of K-ras 4A mRNA normalized to GAPDH mRNA
were 0.45 ± 0.16 and 0.67 ± 0.28 for cell lines with wildtype or
mutant K-ras, respectively (P = 0.55). These values for K-ras 4B
mRNA were 1.22 ± 0.19 and 1.31 ± 0.60, respectively. Thus, for both
4A and 4B, mRNA levels were independent of mutational status.
These real-time mRNA determinations provided relative rather
than absolute values, so it was not possible directly to compare
quantitatively the levels of K-ras 4A mRNA with levels of K-ras 4B.

R.J. Calvert et al. / Lung Cancer 80 (2013) 263–269

267

Fig. 5. (A) Levels of K-ras 4A mRNA relative to GAPDH. Sample size (N) values for K-ras 4A determinations were as follows: HPL, N = 13; H441, N = 12; H1395, N = 4; H1944,
N = 4; H2126, N = 4; A549, N = 3. **P < 0.001 vs. HPL cells, Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. *P < 0.05 vs. HPL, Mann–Whitney pair-wise test.
(B) Significant correlation between K-ras 4A protein and mRNA in cell lines with mutant K-ras. (C) Lack of correlation between K-ras 4A protein and mRNA in cell lines with
wildtype K-ras. (D) Significant correlation between K-ras 4A mRNA and superoxide for all cell lines (P values on graph), including those with mutant K-ras (symbol triangle)
(P = 0.0013) and those with wildtype K-ras (symbol square) (P < 0.0001).

However, for several cancer cell lines it was possible to normalize
4A and 4B mRNAs relative to these values in the non-transformed
HPL line. For H441 cells, 4A mRNA was 70% higher than 4B mRNA
(P = 0.0028), but H1944, H1395, and H2126 did not present significant differences (not shown). Relative total K-ras protein levels
in the cell lines were reported previously [9]. The ratio of relative
K-ras 4A protein to relative total K-ras protein for H441 cells was
0.1, compared to a ratio of 0.002 for HPL cells. Thus in the H441
cell line, at least, the relatively greater 4A mRNA compared with 4B
mRNA corresponded with a relatively greater percentage of total
K-ras protein as 4A protein.

Fig. 6. Levels of K-ras 4B mRNA relative to GAPDH. Sample size (N) values for K-ras
4A determinations were as follows: HPL, N = 10; H441, N = 10; H1395, N = 2; H1944,
N = 3; H2126, N = 3; A549, N = 3. **P < 0.01 vs. HPL cells, Kruskal–Wallis test, followed
by Dunn’s multiple comparisons test. *P < 0.05 vs. HPL, Mann–Whitney pair-wise
test.

4. Discussion
Here we present the first extensive quantification of K-ras 4A
protein in lung cancer cell lines. The amounts of this protein varied 250-fold among the lines. On average, neither 4A protein nor
4A mRNA was significantly different in amount between lines with
wild-type or mutant K-ras. However, there was a significant correlation between 4A protein and mRNA only in cell lines with mutant
K-ras.
In pursuit of an earlier observation linking K-ras activity to
superoxide [9], we looked for a correlation between K-ras 4A and
superoxide levels. Heretofore no report has considered the K-ras
splice variants in the context of reactive oxygen species. We were
not surprised to observe a correlation between K-ras 4A protein
and superoxide levels in lung cancer cell lines containing mutant
K-ras. Unexpected, however, was the experimental evidence, from
manipulation of K-ras and superoxide levels, that mutant K-ras 4A
was not causing superoxide elevation, but rather that superoxide
seemed to be influencing expression of mutant K-ras 4A protein.
We used Tiron to reduce cellular superoxide levels. Tiron could
possibly have unexpected off target effects which might reduce the
level of K-ras 4A protein. In addition to scavenging superoxide,
Tiron also binds metals including iron, copper, uranium, strontium, vanadium, beryllium and chromium [11]. Iron, copper and
chromium would be expected to be present in the adenocarcinoma
cells, while the other metals would need to be deliberately added.
Iron has been reported to facilitate K-ras mutations in naked DNA
through production of hydroxyl radicals [12]. Tiron binding to free
iron might be expected to reduce K-ras mutations in their system
by inhibiting the Fenton reaction. However, alteration of K-ras 4A
protein or mRNA levels associated with reduced iron, copper or

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chromium levels has not been reported. Considering what is known
about Tiron, we think an off-target effect resulting in reduced levels
of K-ras 4A protein is unlikely.
Increased expression of Ras genes in cells has caused increases
in reactive oxygen species. While most studies have utilized Hras (e.g. [13]), K-rasV12 transfected into NRK kidney cells resulted
in upregulation of Nox1 and superoxide [14]. On the other hand,
transfection of the E10 murine lung cell line with K-rasV12 did
not result in increased levels of superoxide compared to wt Kras transfectants or to the parental E10 cell line [15], although
increased peroxides did result, via induction of cyclooxygenase
2.
We sought further understanding of the mutant K-ras 4A
protein–superoxide relationship by testing for correlations. Such
correlations do not, of course, establish cause–effect, but can lead
to speculations and suggestions for further studies. Superoxide correlated with mRNA for K-ras 4A and 4B, both wildtype and mutant.
This suggested superoxide effects on mRNA transcription or stability prior to splicing. Ras genes are among those that respond
immediately to ionizing radiation, which entails superoxide generation [16]. The let-7 family of microRNAs changed expression
in response to ionizing radiation [17]. Let-7 microRNA negatively
regulates Ras gene transcription [18], including K-ras in A549 cells
[19], and Let-7 microRNA reduced growth of mouse lung tumors
[20].
In addition to the putative general effects of superoxide on K-ras
mRNA, there appeared to be possible actions specific to the mutant
protein form, at least mutant K-ras 4A. Wild-type and mutant K-ras
p21s are degraded after intracellular trafficking to lysosomes [21]
with a rate that may be influenced by motility and residence time
in the cell membrane [22,23] and may be greater for the mutant
form [23,24]. This has been shown for K-ras 4B p21 [25,26]; K-ras
4A may not have been specifically studied. Superoxide could affect
these processes indirectly via signaling pathways. Direct effects are
also possible, altering conformation of mutant protein specifically,
to result in increased cell membrane residence or slower trafficking
into the lysosomes. The Cys118 in Ras is redox-active [27].
5. Conclusion
Lung cancer lines with mutant K-ras differed from those with
wildtype K-ras, in that K-ras 4A protein correlated with superoxide
and with K-ras 4A mRNA. Several experiments indicated that superoxide was impacting K-ras 4A protein levels, and not vice versa.
However, superoxide correlated with both 4A and 4B mRNA, in
wildtype as well as mutant cell lines. The results are most consistent
with two different cumulative effects of superoxide: stimulation of
transcription of the K-ras gene; and selective stabilization of K-ras
4A mutant protein.
Superoxide-mediated increases in K-ras 4A mutant protein in
lung cancer cells could be of interest in the contexts of both lung
cancer prevention and treatment, in view of the likely importance
of this isoform for this cancer type. Mitochondrially generated ROS
were needed for full tumorigenicity of a K-ras mutant in mouse lung
[28] and for K-ras 4A activation related to malignant progression
of several cancer types [29].
Conflict of interest statement
None declared.
Acknowledgements
We wish to thank Drs. A. Masuda and T. Takahashi for providing the HPL cells on a collaborative basis. The authors also wish

to recognize the technical assistance of William T. Calvert. This
research was supported by the Intramural Research Program of the
National Institutes of Health, National Cancer Institute, and with
Federal funds from the National Cancer Institute under Contract
HHSN261200800001E. Any opinions, findings, conclusions, or recommendations in this publication are those of the authors and do
not necessarily reflect the views of the Department of Health and
Human Services.

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