Mutant BRAF induces DNA strand breaks,
activates DNA damage response pathway and
upregulates glucose transporter-1 in
nontransformed epithelial cells
Jim Jinn-Chyuan Sheu,* Bin Guan,╪ Fuu-Jen Tsai,* Erin Yi-Ting Hsiao,* Chih-Mei Chen,* Tian-Li Wang,╪ Ie-Ming Shih╪
From the Human Genetic Center,* China Medical University Hospital, Taichung, Taiwan;
Departments of Pathology, Oncology, Gynecology and Obstetrics,╪ Johns Hopkins Medical Institutions, Baltimore, Maryland, USA
Address reprint requests to Ie-Ming Shih, M.D., Ph.D., Cancer Research Building-2, Room 305,1550 Orleans Street, Johns Hopkins Medical Institutions, Baltimore, MD 21231; and to Jim Jinn-Chyuan Sheu, Ph.D., the Human Genetic Center, 2 Yuh-Der Road, China Medical
University Hospital, Taichung, 40447, Taiwan. E-mail: [email protected] and [email protected].
Abstract
While the oncogenic functions of activating BRAF mutations have been well
demonstrated in human cancer, their roles in non-transformed epithelial cells remain largely unclear. Investigating the cellular response to the expression of mutant BRAF in non-transformed epithelial cells is fundamental to understand the roles of BRAF in cancer pathogenesis. In this study, we used two non-transformed cyst108 and RK3E epithelial cells line as the models to compare the phenotypes in cells expressing BRAFwt and BRAFV600E. We found that transfection of the BRAFV600E but not BRAFwt expression vector suppressed cellular proliferation and induced apoptosis in both cell types.
BRAFV600E generated reactive oxygen species, induced DNA double strand breaks and subsequent DNA damage response as evidenced by an increased number of pCHK2 and γH2AX nuclear foci and upregulation of pCHK2, p53 and p21. Since BRAF or KRAS mutations have been correlated with upregulation of GLUT1 that encodes glucose transporter-1, we demonstrated here that expression of BRAFV600E but not with BRAFwt was sufficient to upregulate GLUT1. Taken together, our findings provide new insights into mutant BRAF induced oncogenic stress which is manifested by DNA damage and growth arrest by activating pCHK2-p53-p21 pathway in nontransformed cells while it also confers tumor promoting phenotypes such as upregulation of GLUT1 that contributes to enhanced glucose metabolism that characterizes tumor cells.
Introduction
BRAF probably represents the most frequently mutated oncogene within the kinase family and
activating point mutation at the hot spot V600E of BRAF has been found in several types of human neoplasms, most frequently in melanoma 1, papillary thyroid carcinoma 2, 3, high-grade malignant astrocytoma 4 and ovarian low-grade serous neoplasms 5. BRAF protein is a
downstream effector of KRAS and participates in the signal transduction of the mitogen activated protein kinase (MAPK) pathway that controls cellular growth, differentiation and survival 6, 7. Dimerization of the BRAF kinase domain with KSR or with other RAF molecules has been recently shown to be central to its activation mechanism 8. Activating mutations in BRAF and KRAS appear to exert equivalent tumor-promoting effects as based on the mutual exclusive
mutation in both genes 5, 9. Constitutive activation of BRAF due to V600E mutation activates MAPK pathway and results in upregulation of several genes with tumor-promoting functions including cyclin D1 10, 11 and targeting BRAF and its downstream effectors has emerged as a new therapeutic strategy for those tumors harboring BRAF mutation 12-16.
Ovarian low-grade serous tumor represents a unique type of ovarian epithelial neoplasm and is distinct from ovarian high-grade serous carcinoma, the conventional type of ovarian cancer, based on their clinical, pathological and molecular features 17, 18. Ovarian low-grade serous tumors include a benign form, serous borderline tumor and the malignant counterpart, low-grade serous carcinoma. Low-grade serous carcinoma develops from serous borderline tumor, which in turn may arise from an ovarian serous cystadenoma. Both ovarian low-grade
serous carcinoma and serous borderline tumor harbor BRAF, KRAS or ERBB2 sequence mutation in more than 50% of cases 5,19-21. Expression of active MAPK was more frequently observed in low-grade serous tumors than in high-grade ovarian serous carcinomas that have rare mutations in either BRAF or KRAS 22. Moreover, BRAF and KRAS mutation status is a useful predictor of sensitivity to MEK inhibition in ovarian cancer 11, 23. Interestingly, BRAF or KRAS mutations can be detected in morphologically normal appearing cyst epithelium that is
adjacent to a serous borderline tumor but not in the cystadenomas without concurrent borderline tumors, suggesting the mutations may occur early during tumor progression of ovarian lowgrade serous tumors 24. Although the oncogenic roles of BRAF mutations have been established in mouse models 25, it remains largely unclear what are the biological effects of BRAF mutations in the very beginning of tumor formation such as in non-transformed epithelial cells. Thus, in this study, we ectopically expressed either BRAFV600E or BRAFwt in nontransformed epithelial cells
isolated from ovarian cystadenoma and RK3E cells, an epithelial cell model frequently used to test the oncogenic effects, to determine the phenotypes in both cell lines. Furthermore, a recent study has demonstrated that BRAF expression is required for the expression of GLUT1 which encodes glucose transporter-1 and glucose deprivation is associated with the development of KRAS pathway mutations in tumor cells 26. Thus, in this study, we also tried to determine if
Materials and Methods
Cell growth assay
Expression vectors including the empty vector, wild type BRAF (BRAFWT), and mutant BRAF (BRAFV600E) were kind gifts from Dr. Raquel Seruca (Institute of Molecular Pathology and
Immunology of the University of Porto, Portugal). To determine the effects of BRAFV600E on
nontransformed epithelial cells, we established the cyst108 cell line which was derived from a benign ovarian serous cystadenoma. The reason to use the epithelial cells from a cystadenoma was because cystadenoma represents the immediate precursor lesion of serous borderline tumor. To establish cyst108, we scraped the epithelial cells directly from a benign serous cystadenoma after incubating a fragment of cystadenoma with 0.5% trypsin and EDTA at 37oC for 15 min. The epithelial cells were then rigorously suspended in order to obtain a single cell population. After overnight culture, the cells were immortalized with SV40 large T antigen and the epithelial cells were enriched using cold trypsin treatments to eliminate stromal cell afterwards. Cyst108 cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum and have been passed for at least 30 passages and they exhibited epithelioid morphology under phase contrast microscopy and expressed epithelial cell markers including cytokeratin 18 and Epi-CAM in >98% of cells. They showed contact inhibition in vitro and were not tumorigenic in nu/nu mice for more than 3 months. In this study, we also included the RK3E cell line because it has been widely used to assess transformation ability of potential oncogenes 27-33. Cells
controls, the empty vector and BRAFWT vector were also transfected into the cells. Cell number
was measured daily for 4 consecutive days using the SYBR green I staining method (Molecular Probes, Eugene, OR). The data was expressed as mean ± SD from five duplicates.
Detection of reactive oxygen species
Cyst108 and RK3E cells were seeded on chamber slides (Nunc, Roskilde, Denmark), and subsequently trsansfected with empty vector, BRAFWT, or BRAFV600E vectors on the second day. Seventy-two hours after transfection, cells were stained with 5μM CellROX Deep Red reagent (Invitrogen) in complete medium for 30 min at 37oC, followed by 3 times of wash with PBS and fixation with 4% formaldehyde. Cell nuclei were counter stained with DAPI (Sigma). ROS positive cells were detected and counted under a fluorescent microscope (excitation wave: 644 nm and emission wave: 665 nm). The data was expressed as mean ± SD from triplicates. DNA strand break assay
DNA strand breaks were quantified using a Comet assay kit (Trevigen, Inc., Gaithersburg, MD) as previously described 33, 34. Briefly, transfected cells were harvested in ice-cold PBS, and the cell number was adjusted at a density of 1×105 cells/mL. Cells were mixed with LMAgarose at 1:10 ratio (v/v) and spread onto the CometSlide immediately. After gel solidification, cells on slides were lysed and DNA in the cells was denatured by using the buffers provided by the kit. Fragmented DNA strands were separated from nuclei by electrophoresis and detected by SYBR Green staining. Percentage of comet-like nuclei (with DNA strand breaks) was counted under fluorescent microscope from five randomly selected high-power fields (40X) with each
approximately containing 100 nuclei. UVC-treated cells at sublethal dose were used as the positive control in this assay.
Immunofluorescence staining
To determine whether BRAFV600E expression resulted in DNA damage response, transfected cells were seeded in chamber slides at a density of 5,000 cells per well. At different time points, cells were fixed with para-formaldehyde and incubated with anti-phospho-CHK2 (pCHK2) antibody (clone ab38461; Abcam, Cambridge, MA) or anti-γH2AX antibody (clone ab11174; Abcam) for 2 hrs followed by rhodamin-conjugated anti-rabbit antibody (Jackson ImmunoResearch Lab., West Grove, PA) and nuclei were counterstained with DAPI (Sigma). Cells transfected with an empty vector or BRAFWT vector were used as controls.
Western blot analysis
Protein lysates from different groups were collected at different time points after gene transfection. Proteins were then separated by SDS-PAGE and transferred onto PVDF
membranes. To determine whether BRAFV600E caused activation of the DNA damage response
pathway, we performed Western blots by hybridizing membranes with antibodies against γH2AX (clone ab11174; Abcam), phosphor-CHK2 (clone ab38461; Abcam), p53 (clone sc-6243; Santa Cruz Biotechnology, Santa Cruz, CA) and p21 (clone sc-6246; Santa Cruz), all of which are involved in the DNA damage response pathway 35, 36 for 2 hrs at room temperature. Antibodies against GAPDH were used as the loading control. To detect GLUT1, the membranes were hybridized with an affinity-purified rabbit anti-GLUT1 polyclonal antibody (Millipore, Bedford,
MA). After three washes with TBST (0.01% Tween 20 in TBS), the membranes were blotted with HRP-conjugated anti-mouse (Pierce, Rockford, IL) or anti-rabbit (Cell signaling Technology, Danvers, MA) antibodies for 1 hr at room temperature. Protein bands were revealed by chemiluminescence (Amersham Biosciences, Arlington Heights, IL).
GLUT1 immunohistochemistry
Paraffin-embedded tissues from 33 cases of ovarian low-grade serous tumors (serous borderline tumors and low-grade serous carcinomas) were obtained from the Department of Pathology at the Johns Hopkins Hospital, Baltimore, Maryland. Acquisition of tissue specimens and clinical information were approved under the regulations of the Institutional Review Board. There were 10 cases of low-grade serous carcinomas which metastasized or disseminated to intraperitoneal soft tissues. For immunohistochemistry, the unstained slides were subjected to antigen retrieval by boiling the slides in citrate buffer, pH 6.0 (Zymed, South San Francisco, CA) for 20 min. After blocking, samples were then stained with an affinity-purified rabbit anti-GLUT1 polyclonal antibody (Millipore, Bedford, MA) at a 1:600 dilution at room temperature for one hour. An EnVision+System peroxidase kit (DAKO, Carpentaria, CA) was used for chromogen development. Immunointensity was independently scored by two investigators based on membrane immunoreactivity and labeled as negative (0), weakly positive (1+), moderately positive (2+), and strongly positive (3+) groups. For discordant cases, a third investigator scored and the final intensity score was determined by the majority scores.
Results
To determine the effect of mutant BRAF on cyst108 and RK3E cells, we transfected both cell lines with constructs that expressed mutant (V600E) and wild-type BRAF and compared the proliferative activity of cells in vitro (Fig. 1A and 1B). As shown in Fig. 1C and 1D, both cyst108 and RK3E cells expressing wild-type BRAF continued growing as in vector control groups. In contrast, the proliferative activity significantly decreased in cyst108 and RK3E cells when they expressed BRAFV600E. One of the explanations for the growth inhibitory effects by mutant BRAF is oncogenic stress that describes growth arrest and cellular senescence as a result of
expression of oncogenes in otherwise normal cells 37, 38. As oncogenic stress is a poorly defined process, we determined if it was directly related to DNA damage response. First, we performed immunofluoresence staining for two representative markers of DNA damage including
phosphorylated check point kinase 2 (pCHK2) and phosphorylated histone 2AX (γH2AX) in cyst108 and RK3E cells. We found that BRAFV600E expressing cells demonstrated an increased
number of nuclear foci of pCHK2 and γH2AX as compared to the cells in the control groups including BRAFwt expressing, vector control and parental cells (Fig. 1E and 1F). Ultraviolet
irradiated cells served as the positive control which induced numerous pCHK2 and γH2AX foci in the nuclei. Western blot analysis further demonstrated a time-dependent increase in protein levels of pCHK2, γH2AX, p53 and p21 in BRAFV600E but not in BRAFwt transfected cyst108 and
RK3E cells (Fig. 2A and 2B).
pathway and subsequently induced growth arrest because of upregulation of p53 and p21 proteins. This observation also suggests that DNA strand breaks occur due to mutant BRAF but not wild-type BRAF expression. To demonstrate if this was the case, we directly visualized the individual cells with DNA strand breaks in cyst108 and RK3E cells. Upon electrophoresis, DNA with double strand breaks migrated out of nuclei forming a comet tail-like structure whereas the non-damaged DNA remained within the nuclei. Fig. 2C and 2D showed a higher percentage of comet-like cells in the BRAFV600E expressing group than in control group transfected with BRAFwt
or vector only as early as 48 hrs after transfection. These findings suggest that ectopic expression of mutant BRAF proteins caused DNA strand breaks, initiated DNA damage response and subsequently upregulated p53 and p21, leading to growth arrest in
non-transformed epithelial cells. Although DNA double strand breaks have several causes, we asked in this study whether expression of mutant BRAFV600E was associated with generation of reactive
oxygen species which were detected by CellROX Deep Red reagent. Both cyst108 and RK3E cells were analyzed and their percentage of positive cells was determined under a fluorescent microscope. As shown in Fig. 3, the percentage of positive cells was significantly higher in cells expressing BRAFV600E than cells expressing BRAFWT or in vector control cells.
Several studies have shown that oncogene-induced DNA damage response serves as a molecular pressure to select tumorigenic clones during cancer development 39-41.To study whether BRAFV600E can also play oncogenic roles in promoting tumor progression, we selected
a low cell density (1000 cells/25 cm2). Two BRAFWT cell clones, WT-1 and WT-2, were also analyzed as controls. Anchorage-independent assay showed that cell clones overexpress either BRAFWT or BRAFV600E formed colonies in soft-agar (Fig. 4A). Constitutive expression of
BRAFV600E conferred MT-1 and MT-2 clones to be highly transformed as evidenced by more colonies (Fig. 4A). Consistent with our previous study 42, Western blot analysis (Fig. 4B) and quantitative PCR (Fig. 4C) confirmed lower expression levels of Arf in BRAFV600E-treated, but not in BRAFWT-treated, clones that accounted for the failure of p53 upregulation.
Previous study demonstrated that BRAF mutation was associated with upregulation of GLUT1 which was responsible for increase of glucose uptake and promotion of cellular survival and growth in cancer cells 26. However, it is not known if mutant BRAF is sufficient to upregulate GLUT1 expression. Therefore, we tested whether BRAF mutation causally contributed to GLUT1 overexpression in our system. Western blot analysis demonstrated expression of GLUT1
significantly increased in cyst108 and RK3E cells expressing BRAFV600E as compared to those
cells expressing BRAFwt36 hrs and 48 hrs after transfection (Fig. 5A and 5C). Similarly,
quantitative real time PCR also demonstrated a significant increase in mRNA levels of GLUT1 in cells expressing BRAFV600E (Fig. 5B and 5D). In order to extrapolate the in vitro finding to human
specimens, we performed immunohistochemistry of GLUT1 on a panel of 33 cases of ovarian low-grade serous tumors including 23 serous borderline tumors and 10 low-grade serous carcinomas and correlated their GLUT1 immunoreactivity and the mutation status of BRAF and KRAS. Consistent with our previous study 5, we found that mutations in BRAF and KRAS were
mutually exclusive and mutations in either one of the genes were detected in 18 (55%) of 33 specimens. Specifically, mutations of BRAF or KRAS were found in 6 (60%) of 10
metastatic/disseminated low-grade serous carcinomas whereas the mutations were recorded in 12 (52%) of 23 serous borderline tumors.
Immunohistochemically, we found that all 33 tumor samples were positive for GLUT1 staining except one serous borderline tumor (case 19) (Fig. 6A). GLUT1 immunoreactivity was only detected in cell membrane and cytoplasm of tumor cells but not in stromal cells. All the tumor specimens harboring either BRAF or KRAS mutations showed GLUT1 immunostaining; however, there was no significance in staining intensity among groups with BRAF mutation, KRAS mutation and wild-type (p> 0.1, Mann Whitney test). All 10 cases of low-grade serous
carcinomas expressed GLUT1 protein of which the immunostaining intensity score ranged from 1 to 3. Representative photomicrographs of GLUT1 staining in three advanced stage low-grade serous carcinomas with different mutation status of BRAF and KRAS were illustrated in Fig. 6B.
Discussion
Using both ovarian cystadenoma epithelial cells and RK3E cells as the models, we were able to demonstrate that mutant BRAF induced growth arrest in both cell types and such “oncogenic stress” is attributed at least in part by DNA damage response pathway that subsequently activate p53 and p21. We provide cogent evidence in this report that expression of mutant but not wild-type BRAF directly causes DNA strand breaks and accounts for the activation of DNA damage response. Transcription-induced double DNA strand breaks have been proposed to occur when novel transcription is induced during tumor development 43. It is likely that expression of mutant BRAF, like deregulated expression of Myc 44, induces oxidative stress that is
responsible for topoisomerase TOP2B-dependent DNA double strand breaks in epithelial cells. In fact, we have also observed that expression of mutant BRAF was associated with generation of reactive oxygen species in epithelial cells. As occurs in oncogenic stress, increased p53 levels due to the ATM-pCHK2-p53-p21 pathway activation lead to cell growth arrest at G1 or G2/M and/or in apoptosis 45. Consistent with view, it has also been reported that ovarian serous borderline tumors have a much lower proliferative activity 46, 47 and a significantly lower TP53 mutation frequency than ovarian high-grade serous carcinoma, the conventional type of ovarian cancer, that harbor neither BRAF or KRAS mutations 48. Although this is our preferred view, other mechanisms for mutant BRAF-induced p53 activation should be also pointed out. For example, a recent study demonstrates that the Jnk pathway signaling is involved in the activation of p53 in response to both KRAS and Neu oncogene expression 49.
The results from this study may help further understanding the molecular pathogenesis of ovarian low-grade serous carcinoma. It can be speculated that epithelial cells from a serous borderline tumor may evolve a mechanism to restrain tumor progression 39-41. In response to BRAF mutations, activation of ATM/pCHK2 /p53/p21 is thus important to suppress tumor cell
proliferation. Although not frequently, serous borderline tumors may progress to low-grade serous carcinomas which are frankly malignant neoplasm and are often associated with high morbidity and mortality. How do tumor cells in low-grade serous carcinoma overcome the growth inhibitory effect due to activating mutations of BRAF? We propose that additional molecular genetic alterations occur during progression from a serous borderline tumor to a low-grade serous carcinoma, and such molecular alterations abolish the checkpoint control by the ATM-p53 pathway, allowing cells to proliferate despite the presence of mutant BRAF-induced DNA damage and upregulation of p53 and p21. To this end, a previous study that analyzed the genome-wide copy number alterations in ovarian serous neoplasms has reported that hemizygous ch1p36 deletion and ch9p21 homozygous or hemizygous deletions were much more common in ovarian low-grade serous carcinomas than in serous borderline tumor 42. The ch1p36 region contains several candidate tumor suppressors including miR-34a which is required for DNA damage response and is the direct p53 target that mediates its tumor suppressor functions 50, 51. Similarly, the ch9p21 region corresponding to the CDKN2A/B locus encodes three well-known tumor suppressor proteins, p14 (Arf), p16 and p15. CDKN2A and CDKN2B share similar function in inhibiting cyclin-dependent kinase. Arf is a potent tumor
suppressor that blocks cell cycle progression by interfering with the p53 negative regulator, MDM2, thereby stabilizing p53 protein expression. Besides, the expression level of CDKN2A was enhanced in response to oncogene-induced stress such as by the activation of
RAS/RAF/MEK signaling pathway. Thus, deletions or silencing of miR-34a and CDKN2A/B loci may uplift the p53 checkpoint upon BRAF mutations and permit tumor cells to escape from cell cycle arrest and become more aggressive, as shown in Fig. 4. The above view is supported by the fact that expression of BRAFV600E in the lung epithelium or in melanocytes fails to result in frankly malignancy unless tumor suppressor genes such as Pten are inactivated 52, 53.
If BRAF mutations result in growth arrest in serous borderline tumor, why this genotype is clonally selected and can be detected in low-grade serous carcinomas even when they are at advanced stages (Fig. 6A)? We reasoned that once tumor cells bypass the oncogene-induced growth arrest, they may benefit from tumor-promoting phenotypes conferred by BRAF mutations such as metabolic switches among several tumor-promoting functions. To explore this
possibility, we focused on GLUT1, a gene that encodes glucose transporter-1, which has been implicated to play a critical role in regulating glucose metabolism and energy consumption in cancer cells 54. The reason to focus on GLUT1 stems from a recent report showing that either BRAF or KRAS mutation is required for GLUT1 overexpression and glucose deprivation
contributes to the development of mutations in BRAF and KRAS in colorectal cancer cells 26. In that report, the glycolysis inhibitor, 3-bromopyruvate, preferentially inhibited the growth of cells with either BRAF or KRAS mutations, suggesting that cancer cells may develop dependency on
increased glucose metabolism due to GLUT1 overexpression. The observation in the current study demonstrating that BRAF mutation induced upregulation of GLUT1 expression, thus, provides direct evidence that mutant BRAF is not only required but also sufficient to upregulate GLUT1 expression.
We also report in this study that the great majority of low-grade ovarian serous tumors express GLUT1 based on immunohistochemistry. More specifically, while all borderline tumors harboring either BRAF or KRAS mutations express GLUT1, those cases with wild-type BRAF and KRAS also show GLUT1 immunoreactivity except in one case. This observation indicates that some low-grade ovarian serous tumors with wild-type BRAF and KRAS upregulate GLUT1 using different mechanism not directly related to mutations of BRAF or KRAS, such as those mediated by hypoxia 55. Our data demonstrate that mutation of BRAF and KRAS represents one of the mechanisms to upregulate GLUT1. It can be speculated that for those tumors with either
BRAF or KRAS mutation, blocking the pathway by MEK or BRAF inhibitors may be responsible
for tumor suppression due to downregulation of GLUT1 expression. For those tumors with wild-type BRAF and KRAS, MEK or BRAF inhibitor may not work well because alternative
mechanisms are used by those tumor cells to upregulate GLUT1. Because expression of GLUT1 has been shown as a reliable marker to predict positive fluorodeoxyglucose uptake by positron emission tomography in ovarian cancer 56, our data suggest the potential to apply
flourodeoxyglucose uptake imaging to detect lowgrade ovarian serous carcinomas.
induced by mutant BRAF in non-transformed epithelial cells. We demonstrate for the first time that expression of mutant but not wild-type BRAF leads to DNA double strand breaks, followed by activation of pCHK2-p53 DNA damage response pathway that is responsible for growth inhibition and tumor suppression. On the other hand, similar to other oncogenes that regulate cellular metabolism in favor of tumor growth 57, mutant BRAF also confers oncogenic phenotypes by upregulating GLUT1 of which abundant GLUT1 proteins contribute to enhanced glucose uptake and metabolism that characterizes cancer cells.
Acknowledgements
The authors are grateful for the technical support from I-Wen Chiu and Carmen Chan at China Medical University Hospital and for the kind gift of BRAF expression vectors from Dr. Raquel Seruca, University of Porto, Portugal. This study is supported by China Medical University (CMU97-001).
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Legends
Fig. 1. Expression of mutant BRAF (V600E) suppresses cellular proliferation and induced pCHK2 and γH2AX nuclear foci. Forty-eight hours after transfecting epithelial cells, robust expression levels of wild-type and mutant BRAF can be detected in cyst108cells (A) and in RK3E cells (B). A reduced cellular proliferation was recorded in cyst108cells (C) and in RK3E cells (D) that expressed mutant BRAF. Forty-eight hours aftertransfection, cells were stained for pCHK2 and γH2AX. Nuclear foci for pCHK2 and γH2AXimmunofluoresence were observed in mutant BRAF expressing cyst108 cells (E) and RK3E cells (F). Ultraviolet light (UV) treated cells serve as the positive controls forimmunofluroresence staining.
Fig. 2. Mutant BRAF activates DNA damage response pathway and induces DNA strandbreaks. Western blot analysis demonstrates a time-dependent increase in protein expression ofp53, pCHK2, γH2AX and p21 in cyst108 cells (A) and in RK3E cells (B). GAPDH serves as the protein loading control. Cells with DNA strand breaks were analyzed by the Comet assay. A significantly higher percentage of comet-like nuclei are found in the BRAFV600E expressing group
than in control group transfected with BRAFwt or vector only 48 hrs after transfection.
Fig. 3. Measurement of reactive oxygen species in cyst108 and RK3E cells. CellROX DeepRed reagent was used to detect the reactive oxygen species and percentage of positive cellswas recorded under a fluorescent microscope. Cell nuclei were counter stained with DAPI (blue
fluroresence). The percentage of positive cells (red fluorescence in cytoplasm) is significantly higher in cells expressing BRAFV600E than cells expressing BRAFWT or vector control cells. A: cyst108 cells; B: RK3E cells.
Fig. 4. Long-term expression of BRAFV600E promotes a more aggressive phenotype in cyst108 cells. Soft agar assay was performed to detect the tumorigenic activity of cell clones that overexpress BRAFV600E or BRAFWT under G418 selection (A). Western blot analysis (B) and quantitative PCR (C) were performed to detect expression levels of BRAF, Arf and p53 in selected cell clones. GAPDH was utilized as the protein loadingcontrol.Vector-treated cells serve as controls in both assays.
Fig. 5. Increased GLUT1 protein expression in cells that express mutant BRAF. Western blot analysis was performed 36 hours and 48 hours after transfection. Expression of GLUT1
significantly increases in cyst108 (A) and RK3E cells (C) expressing BRAFV600E as compared to
those cells expressing BRAFwt at both time points. GAPDH serves as the protein loadingcontrol. Quantitative PCR was performed to detect mRNA levels of GLUT1 in cyst108 (B) and RK3E cells (D) 48 hours after gene transfection.
Fig. 6. Expression of GLUT1 in low-grade ovarian serous tumors and the correlation of GLUT1 immunoreactivity with mutation status of BRAF and KRAS. Immunohistochemistry was
performed in 33 cases of low-grade ovarian serous tumors including 23 serous borderline tumors and 10 advanced stage low-grade serous carcinomas. The GLUT1 immunostaining intensity is shown for all cases (A) which are grouped into BRAF mutation, KRAS mutation and wild-type groups. Mutations of KRAS and BRAF are mutually exclusive. Low-grade serous carcinomas are labeled with asterisks otherwise they are serous borderline tumors.
Representative photomicrographs of low-grade serous carcinomas from each group are illustrated and their mutation status in KRAS and BRAF is indicated below. The case numbers correspond to those shown above.
