research article the rassf1 gene and the opposing effects

Hindawi Publishing CorporationMolecular Biology InternationalVolume 2013 Article ID 145096 9 pageshttpdxdoiorg1011552013145096

Research ArticleThe RASSF1 Gene and the Opposing Effects of the RASSF1A andRASSF1C Isoforms on Cell Proliferation and Apoptosis

Mark E Reeves12 Matthew Firek1 Shin-Tai Chen3 and Yousef Amaar12

1 Surgical Oncology Laboratory Loma Linda VA Medical Center Loma Linda CA 92357 USA2Department of Surgery Loma Linda University Loma Linda CA 92357 USA3Musculoskeletal Disease Center Loma Linda VA Medical Center Loma Linda CA 92357 USA

Correspondence should be addressed to Yousef Amaar yousefamaarvagov

Received 22 July 2013 Accepted 25 September 2013

Academic Editor Emanuel Strehler

Copyright copy 2013 Mark E Reeves et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

RASSF1Ahas been demonstrated to be a tumor suppressor while RASSF1C is now emerging as a growth promoting protein in breastand lung cancer cells To further highlight the dual functionality of the RASSF1 gene we have compared the effects of RASSF1Aand RASSF1C on cell proliferation and apoptosis in the presence of TNF-120572 Overexpression of RASSF1C in breast and lung cancercells reduced the effects of TNF-120572 on cell proliferation apoptosis and MST12 phosphorylation while overexpression of RASSF1Ahad the opposite effect We also assessed the expression of RASSF1A and RASSF1C in breast and lung tumor and matched normaltissues We found that RASSF1A mRNA levels are significantly higher than RASSF1C mRNA levels in all normal breast and lungtissues examined In addition RASSF1A expression is significantly downregulated in 92 of breast tumors and in 53 of lungtumors Conversely RASSF1C was upregulated in 62 of breast tumors and in 47 of lung tumors Together these findings suggestthat RASSF1C unlike RASSF1A is not a tumor suppressor but instead may play a role in stimulating survival in breast and lungcancer cells

1 Introduction

RASSF1A and RASSF1C are the two major isoforms encodedby the RASSF1 gene RASSF1A is a key tumor suppressorIt is inactivated in the vast majority of human cancersincluding breast and lung cancers [1ndash5] Recently we andothers demonstrated that the RASSF1C isoform promotescell proliferation and migration and attenuates apoptosis inbreast and lung cancers [6ndash10] This suggests that RASSF1Cand RASSF1A have opposing effects on cancer cells Thisis further supported by recent cohort studies in humanlung and pancreatic cancers showing that elevated RASSF1Cexpression correlates significantly with poor patient survival[11 12]

To shed more light on this newly emerging conceptof RASSF1 dual functionality we have undertaken studieswhich compare the impact of the RASSF1A and RASSF1Cisoforms on TNF-120572-induced apoptosis RASSF1A is inti-mately involved in the Hippo and MOAP1Bax proapoptotic

pathways RASSF1A promotes apoptosis through activationof both MST kinases in the Hippo pathway and activationof Bax in the MOAPBax pathway through TNF-120572 receptoractivation RASSF1A promotes and maintains phosphoryla-tion of MST1 and also activates Bax leading to the induc-tionactivation of downstream of proapoptotic genes such aspuma and caspases 3 and 7 genes [13ndash17] Like RASSF1ARASSF1C is able to bind MST12 proteins As such we rea-soned that RASSF1C could potentially interfere with MST12activation and antagonize the effects of RASSF1A Thereforewe used the TNF-120572-induced apoptosis system to compareand contrast the effects of RASSF1A and RASSF1C in breastand lung cancer cells To do this we overexpressed RASSF1Aand RASSF1C in both breast and lung cancer cells We thenused these cells to first assess cell proliferation and then tomeasure caspase 37 activation and MST phosphorylation inthe presence of TNF-120572 Lastly we also assessed the expressionof RASSF1A and RASSF1C in breast and lung tumor andnormal tissues

2 Molecular Biology International

2 Materials and Methods

21 Cell Culture The human breast cancer cell line T47Dand lung cancer cell line NCI-H1299 stably overexpressingHA-RASSF1A or HA-RASSF1C were developed as previouslydescribed [6] Cells overexpressing HA-RASSF1A (T47D-1A H1299-1A) HA-RASSF1C (T47D-1C H1299-1C) or theempty MLV backbone (T47D-BB H1299-BB) were grownin RPMI-1640 medium supplemented with 10 calf bovineserum For growth of T47D cells mediumwas supplementedwith 02UmL insulin

22 Western Blot Analysis Western blot analysis was carriedout using standard procedures with detection on the OdysseyInfrared System (LI-COR Biosciences Lincoln NE) Rabbitpolyclonal anti-MST12 and anti-pMST12 (Cell Signaling)HA-tag antibody (Covance) anti-YAP and anti-pYAP (CellSignaling) and goat anti-mouse and donkey anti-rabbitfluorescently labeled secondary antibodies (IRDye 680 926-32220 LI-COR Biosciences) were used

23 Treatment of Cells with TNF-120572 Cells were plated in 96-well plates at 5000 cellswell and were treated the next daywith 1 120583gmL Actinomycin D for 2 hr Cells were then treatedwith 40 120583M TNF-120572 in DMSO (Sigma St Louis MO) for6 hr and then analyzed by western blot Alamar Blue cellviability assays were performed 18 hr after TNF-120572 treatmentas previously described [7] Cell proliferation was measuredby the Alamar Blue assay as previously described [6 7]Data are presented as mean values plusmn SEM and analyzed withStudentrsquos 119905-test Values le005 were considered significant

24 Caspase 37 Activity Assay Cells treated with TNF-120572 for6 hr were assayed for caspase 37 activity using the Apo37caspase activity assay (Promega Madison WI)

25 RNA Isolation and RT-PCR Analysis Tumor andmatched normal tissues were obtained from the WesternDivision of the Cooperative Human Tissue Bank (VanderbiltUniversity TN)

Tumor and normal breast and lung tissues were groundto a powder using a mortar and pestle and the tissuepowder was used to isolate total RNA using PureLink RNAMini Kit (Invitrogen Carlsbad CA) 1120583g of total RNAwas used in reverse transcriptase (RT) reactions using thesuperscript kit (Qiagen Germantown MD) and the RTreactions were subsequently used to set up real-time PCR(RT-PCR) reactions using 1 120583L of RT as a template The RT-PCR reactions were set up using SYBR green PCR mastermix (BioRad Hercules CA) and the PCR reactions wererun using the Opticon 2 PCRmachine (BioRad) Cyclophilinwas used as a loading control Expression of RASSF1A andRASSF1C was assessed by qRT-PCR using RASSF1A andRASSF1C specific primersThe PCR reactions were run usingthe following protocol (1) incubate at 95∘C for 10min (2)incubate at 95∘C for 15 sec (3) incubate for 30 sec (4) go toline 2 for 39 more cycles (5) melt curve from 60∘C to 95∘Cread every 10∘C and (6) incubate at 10∘C forever qRT-PCR

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Figure 1 Breast T47D and lung NCI-H1299 cancer cells sta-bly transduced with MLV backbone or MLV vectors contain-ing HA-RASSF1A or HA-RASSF1C overexpress the transducedprotein as judged by western blots using HA-antibody T47D-BB=T47D transducedwithMLVbackbone T47D-1A=T47D over-expressing HA-RASSF1A T47D-1C=T47D overexpressing HA-RASSF1CH1299-BB=NCI-H1299 transducedwithMLVbackboneand H1299-1A=NCI-H1299 overexpressing HA-RASSF1A H1299-1C =NCI-H1299 overexpressing HA-RASSF1C

reactions were carried out in triplicate and the fold changewas calculated using the 2minusΔΔCT method [18] The RASSF1Aforward primer is 51015840 GGCGTCGTGCGCAAAGGCC 31015840the RASFF1C forward primer is 51015840 CTGCAGCCAAGAG-GACTCGG 31015840 and the reverse primer for both RASSF1Aand RASSF1C is 51015840 GGGTGGCTTCTTGCTGGAGGG 31015840The cyclophilin (housekeeping gene) forward primer is51015840 GCATACAGGTCCTGGCATCT 31015840 and the cyclophilinreverse primer is 51015840 TCTTGCTGGTCTTGCCATTC 31015840 PCRreactions were set in triplicates and cyclophilin was used asan internal loading control and was used to normalize therelative expression levels using the 2minusΔΔCT method [18]

3 Results

31 RASSF1C Reduces the Effects of TNF-120572 on Cell Prolifera-tion TNF-120572 induces cell apoptosis and it has been demon-strated that RASSF1A mediates the apoptotic effects of TNF-120572 In contrast we have previously shown that overexpressionof RASSF1C attenuates apoptosis in breast and lung cancercells In this study we compared the effects of RASSF1Aand RASSF1C overexpression on breast and lung cancer cellproliferation in the presence and absence of TNF-120572 (Figures1 2 and 3) We found that while RASSF1A reduced cell pro-liferation and further enhanced the antiproliferative effectsof TNF-120572 RASSF1C significantly increased cell proliferationand reduced the antiproliferative effects of TNF-120572 on breastand lung cancer cells Our findings suggest that RASSF1A andRASSF1C have antagonistic effects on cell proliferation

32 RASSF1C Reduces Caspase 37 Activation in Breast andLung Cancer Cells Treated with TNF-120572 To further investigatethe antagonism of the antiproliferative effects of TNF-120572 byRASSF1C we assessed the activation of caspase 37 in breastand lung cancer cells overexpressing RASSF1A and RASSF1Cthat were treated with TNF-120572 Both breast and lung cancercells overexpressing RASSF1C showed a significant reductionin caspase 37 activation while RASSF1A overexpressionenhanced caspase 37 activity (Figure 4) This suggests thatRASSF1C has antiapoptotic effects on breast and lung cancercells and further supports antagonistic effects of RASSF1Aand RASSF1C on apoptosis

Molecular Biology International 3

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Figure 2 T47D breast (a) and H1299 lung (b) cancer cells stably overexpressing RASSF1A or RASSF1C were assayed for cellviabilityproliferation in absence of TNF-120572 using the Alamar Blue assay RASSF1A overexpression significantly reduced while RASSF1Coverexpression significantly enhanced cell viabilityproliferation compared to control (BB) cells Data is representative of at least 3independent experiments and the values represent the meanplusmn SEM lowast119875 lt 005 for 1C compared to 1A and BB and for 1A compared toBB

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Figure 3 T47D breast (a) and NCI-H1299 lung (b) cancer cells overexpressing RASSF1A or RASSF1C were treated with TNF-120572 for 24 hrand then cells were assayed for cell viabilityproliferation RASSF1A overexpression significantly enhanced while RASSF1C overexpressionsignificantly reduced the sensitivity of cells to TNF-120572 Data is representative of at least 3 independent experiments and the values representthe meanplusmn SEM lowast119875 lt 005 for 1C compared to 1A and BB and for 1A compared to BB

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Figure 4 Caspase 37 activation was assessed in breast T47D (a) and lung H1299 (b) cancer cells overexpressing RASSF1A or RASSF1C afterTNF-120572 treatment for 24 hr Overexpression of RASSF1C significantly reduced caspase 37 activation in both breast and lung cancer cells Datais representative of at least 3 independent experiments and the values represent the meanplusmn SEM lowast119875 lt 005 for 1C compared to 1A and BBand for 1A compared to BB


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Figure 5 (a) These breast or lung cancer cells overexpressing RASSF1A or RASSF1C were treated with TNF-120572 and cell lysates were used toassess MST1 phosphorylation RASSF1A overexpression increased pMST in both breast and lung cancer cells and RASSF1C overexpressionslightly reduced pMST in breast cancer cells but had no effect on pMST in lung cancer cells treated with TNF-120572 (b) Overexpression ofRASSF1C appears to reduce and RASSF1A appears to increase total MST levels compared to control The blots shown represent one of threeindependent experiments

33 RASSF1C Overexpression Effects on MST12 or YAPPhosphorylation In order to help understand how RASSF1Aand RASSF1Cmight exhibit antagonistic effects on apoptosiswe studied the effects of overexpression of RASSF1A andRASSF1C on the Hippo pathway While RASSF1A promotesapoptosis through the Hippo pathway by inducing mam-malian sterile 20-like kinases 1 and 2 (MST12) phosphory-lation nothing is known about the impact of RASSF1C onMST12 phosphorylation or its effect on the Hippo pathwaymore broadly Therefore we assessed the phosphorylationof MST12 in breast and lung cancer cells overexpressing

RASSF1A and RASSF1C in the presence of TNF-120572 (Figure 5)We found that in breast cancer cells overexpression ofRASSF1A enhanced MST12 phosphorylation as expectedbut RASSF1C overexpression slightly reduced it (Figure 5(a))However in lung cancer cells while RASSF1A also enhancedMST12 phosphorylation overexpression of RASSF1C hadno effect on MST12 phosphorylation (Figure 5(a)) We alsoassessed the levels of total MST12 in breast and lung cancercells overexpressing RASSF1A or RASSF1C and found thatRASSF1A appears to enhance andRASSF1C appears to reduceMST12 protein levels compared to control (Figure 5(b))


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Figure 6 Breast T47D and lung NCI-H1299 cancer cells stably overexpressing RASSF1A or RASSF1C were treated with TNF-120572 and celllysates were used to assess YAP phosphorylation (pYAP) (a) Breast cancer cells overexpressing RASSF1A showed a small increase in pYAPcompared to cells overexpressing RASSF1C but cells overexpressing RASSF1A or RASSF1C showed reduced pYAP compared to control (BB)Overexpression of RASSF1A reduced and RASSF1C increased total YAP in the presence of TNF-120572 in breast cancer cells (b) Overexpressionof RASSF1A slightly reduced and RASSF1C overexpression slightly increased pYAP protein levels in the presence of TNF-120572 in lung cancercells but pYAP levels in cells overexpressing RASSF1A or RASSF1C are lower compared to those in control cells Overexpression of RASSF1Aslightly reduced and RASSF1C significantly decreased total YAP protein levels in the presence of TNF-120572 compared to those in control cellsThe blots shown represent one of three independent experiments

We also assessed the phosphorylation of the Yorkieassociated protein (YAP) in the presence of TNF-120572 YAP isa downstream effector protein in the Hippo pathway Breastcancer cells overexpressing RASSF1A or RASSF1C exhibitedan increase in phosphorylated YAP levels However breastcancer cells overexpressing RASSF1A had slightly decreasedlevels of total YAP while cells overexpressing RASSF1C hadsignificantly increased levels of total YAP (Figure 6(a)) Inlung cancer cells RASSF1A overexpression slightly reducedboth pYAP and total YAP levels while RASSF1C overexpres-sion slightly increased pYAP and significantly reduced total

YAP levels compared to untreated cells (Figure 6(b))Thus itis possible that the antiproliferative and proapoptotic effectsof TNF-120572may not involve YAP phosphorylation

34 RASSF1A and RASSF1C Expression in Normal Breastand Lung Tissues The balance of the ratio of expressionof RASSF1 isoforms relative to each other may be criticalfor maintaining normal versus transformed growth Hencethe expression of RASSF1A and RASSF1C was assessed innormal breast and lung tissues using RT-PCR (Figure 7)


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Figure 7 Assessment of RASSF1A and RASSF1CmRNA expression was carried out in normal breast ((a) 3 samples) and normal lung tissues((b) 15 samples) using RT-PCR analysis Data shows that RASSF1A mRNA levels are significantly higher than RASSF1C mRNA levels innormal breast and lung tissues PCR reactions were set in triplicate and cyclophilin was used as an internal loading control and was used tonormalize the relative expression levels using the 2minusΔΔCT method [18]

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Figure 8 RASSF1A and RASSF1C mRNA were assessed in 13 breast tumor tissues and normal tissues using RT-PCR (a) RASSF1A isdownregulated in 92 (1213) and is upregulated in 8 (113) and (b) RASSF1C is downregulated in 15 and is upregulated in 62 (813) ofthe breast tumors assessed 1A N relative RASSF1A expression in normal breast tissue 1A T relative RASSF1A expression in breast tumortissue 1C N relative RASSF1C expression in normal breast tissue 1C T relative RASSF1C expression in breast tumor tissue

Interestingly RASSF1A mRNA levels are significantly higherthan RASSF1C mRNA levels in three normal breast tissuesand the overwhelming majority of 15 normal lung tissuesexamined This is consistent with the concept that higherRASSF1A expression levels relative to RASSF1C levels maycontribute to normal cellular growth and prevent trans-formed cellular growth

35 RASSF1A and RASSF1C Expression in Tumor TissueWe also assessed the expression of RASSF1A and RASSF1CmRNA levels in tumor and matched normal breast andlung tissues Consistent with previously reported resultsRASSF1A expression was significantly downregulated in 92of breast tumors and 53 of lung adenocarcinomas (Figures

8 and 9) In contrast RASSF1C expression was significantlyupregulated in 62 of breast tumors and in 47 of lungadenocarcinomas (Figures 8 and 9) This further suggeststhat unlike RASSF1A RASSF1C is not a tumor suppressorRather RASSF1C may actually have a growth promotingfunction in tumors Consistent with this we found that theratio of RASSF1C to RASSF1A is greater than 1 in 100 ofbreast and 60 of lung tumors (Figure 9) Thus it is possiblethat the expression ratio of RASSF1 isoforms is important forsupporting normal versus oncogenic growth behavior

4 Discussion

Evidence is accumulating to support the hypothesis thatthe RASSF1 gene may function to balance critical cellular


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Figure 9 Assessment of RASSF1A and RASSF1CmRNA expression was carried out in 15 lung adenocarcinomas andmatched normal tissuesusing RT-PCR (a) RASSF1A was downregulated in 53 (815) and upregulated in 20 (315) and (b) RASSF1C was downregulated in 53(815) and upregulated in 47 (715) of the lung adenocarcinomas tested

functions that can tip the balance between normal andneoplastic behavior depending on whether the RASSF1Aor RASSF1C isoform is predominantly expressed [6ndash12]RASSF1A functions as a tumor suppressor by inhibiting cellproliferation and migration and promoting apoptosis [4 513] In contrast we and others have shown that RASSF1C hasan opposite effect promoting cancer cell proliferation andmigration and attenuating apoptosis in breast and lung cancercells [6ndash10] RASSF1A mediates the apoptotic effects of TNF-120572 through the activation of modulator of apoptosis protein 1(MOAP1) and Bax leading to caspase 3 activation [13] TNF-120572is an inflammatory cytokine that regulates cell proliferationand cell death and resistance to TNF-120572-induced apoptosismay lead to tumor formation and growth In this studywe have conducted studies directly comparing the effects ofRASSF1A and RASSF1C on breast and lung cancer cell prolif-eration and apoptosis in the presence and absence of TNF-120572We confirmed that overexpression of RASSF1A reducedwhileoverexpression of RASSF1C enhanced breast and lung cancerproliferationviability (Figure 2) More importantly our datashow that RASSF1C reduced while RASSF1A enhanced theantiproliferative effects of TNF-120572 on breast and lung cancercells This adds further evidence for growth-promoting andapoptosis-attenuating functions for RASSF1C Our currentfindings are consistentwith our previously reportedwork thatRASSF1C reduces the sensitivity of breast cancer cells to thechemotherapeutic agent etoposide [8] More recent studiesshow that RASSF1A overexpression induced caspase 3 geneexpression and activity in melanomas [19] Consistent withthis we found that caspase 37 activation was significantlyincreased in breast and lung cancer cells overexpressingRASSF1A which were treated with TNF-120572 compared to thoseoverexpressing RASSF1C (Figure 4) We have previouslyshown that RASSF1C down-regulates caspase 3 expressionand activity in breast cancer cells [8] and now in lungcancer cells as well Thus our results show that RASSF1A

and RASSF1C have opposite effects on caspase 3 both atthe transcriptional and at the protein activation levels Thisfurther supports a dual role of the RASSF1 gene

Recently RASSF1A has been demonstrated to interactwith MST12 through the SalvadorRassfHippo (SARAH)domain to activate apoptosis by relieving Raf1 inhibition ofthe MST12 signaling pathway eventually leading to H2Bphosphorylation and DNA fragmentation Thus we assessedMST12 phosphorylation in breast and lung cancer cellsoverexpressing RASSF1A or RASSF1C We found that whileRASSF1A overexpression induced MST12 phosphorylationRASSF1C overexpression attenuated MST12 phosphoryla-tion in breast and lung cancer cells after TNF-120572 treatment(Figure 5) Also overexpression of RASSF1C appears toreduce and overexpression of RASSF1A appears to increasethe total MST protein levels (Figure 5(b)) This demon-strates another functional difference between RASSF1A andRASSF1C that may well affect apoptosis Since both RASSF1Aand RASSF1C contain the SARAH domain located in theiridentical C-termini RASSF1C should be capable of interact-ing with the SARAH domain-containing proteins and couldpotentially attenuate MST12-mediated apoptosis We havealso assessed the phosphorylation status of the YAP proteina downstream effector of the Hippo pathway Breast and lungcancer cells overexpressing RASSF1A showed slightly higherlevels of phosphorylated YAP compared to those cells over-expressing RASSF1C Interestingly levels of phosphorylatedYAP were reduced in the same breast and lung cancer cellsoverexpressingRASSF1Aor RASSF1C and in lung cancer cellsoverexpressing RASSF1C upon treatment with TNF-120572 Sinceboth RASSF1A and RASSF1C contain the SARAH domainlocated in their identical C-termini RASSF1C should becapable of interacting with the SARAH domain-containingproteins and could potentially attenuate MST12-mediatedapoptosis More focused and detailed studies investigatingthe impact of RASSF1C on the Hippo and the MOAP1Bax


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Figure 10 The ratio of RASSF1C to RASSF1A expression was assessed in breast and lung tumor tissues The ratio of RASSF1C to RASSF1Awas greater than 1 in 100 of breast (a) and 60 of lung (b) tumors The ratio was calculated by dividing (RASSF1C expression in tumortissueRASSF1C in normal tissue) by (RASSF1A expression in tumor tissueRASSF1A in normal tissue)

proapoptotic pathways are needed and we are undertakingthese studies

In this paper we also assessed the expression profileof RASSF1A and RASSF1C in tumor and matched normaltissues using RT-PCR analysis Published work demonstratesthat RASSF1A is a tumor suppressor and its expression isdownregulated in many different types of human cancersincluding breast and lung [1ndash5] In contrast RASSF1C is wellexpressed in most human solid tumors including breast andlung cancers It has been suggested that the expression ratioof RASSF1A to RASSF1C may be critical when it comes tomaintaining normal growth versus inducing oncogenic cellgrowthTherefore we assessed the mRNA levels of RASSF1Aand RASSF1C in normal breast and lung tissues using RT-PCR We found that RASSF1A mRNA levels are consistentlyand significantly higher than RASSF1C mRNA levels in allnormal breast and lung tissues examined (Figure 7) It ispossible that a higher RASSF1ARASSF1C expression ratiowould suppress the oncogenic growth effects of RASSF1Cand RASSF1A functions could be dominant However incancers the oppositemay be trueWhenRASSF1A expressionis downregulated due to methylation in cancer cells theRASSF1CRASSF1A expression ratio would allow for thegrowth-promoting and antiapoptotic actions of RASSF1CConsistent with this we found that the ratio of RASSF1Cto RASSF1A is elevated in 100 of breast and 60 of lungtumors (Figure 10) Whether the increase of RASSF1C func-tion is mainly due to a decreased RASSF1A expression aloneor is due to a combination of suppression of RASSF1A expres-sion and up-regulation of RASSF1C expression remains to bethoroughly investigated

Previous studies have shown that RASSF1C expressionis significantly upregulated and RASSF1A is significantlydownregulated in lung and pancreatic tumor tissues and thatup-regulation of RASSF1C correlates with poor prognosisand survival [11 12] Consistent with this we also found thatRASSF1A expression is significantly downregulated in 92 ofbreast tumors and 53 of lung tumors On the other handRASSF1C was upregulated in 62 of breast tumors and 47

of lung tumors Our findings show that RASSF1C expressionis significantly elevated in half of breast and lung cancersfurther supporting a growth promoting role for RASSF1C inthese cancers Obviously larger tissue sample sizes of breastcancer lung cancer and other cancer types of cancerwill needto be examined

5 Conclusions

Our findings suggest that RASSF1C has opposite effects oncell proliferation and apoptosis in breast and lung cancercells compared to RASSF1A Our current and previouslypublished data suggest that RASSF1C can function as agrowth promoting protein and further support the emergingconcept of the dual functionality of the RASSF1 gene

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was carried out at the Loma Linda VA MedicalCenter Loma Linda CA The work was supported in part bythe Loma Linda University Cancer Center

References

[1] R Dammann C Li J H Yoon P L Chin S Bates and G PPfeifer ldquoEpigenetic inactivation of a RAS association domainfamily protein from the lung tumour suppressor locus 3p213rdquoNature Genetics vol 25 no 3 pp 315ndash319 2000

[2] DG Burbee E Forgacs S Zochbauer-Muller et al ldquoEpigeneticinactivation of RASSF1A in lung and breast cancers and malig-nant phenotype suppressionrdquo Journal of the National CancerInstitute vol 93 no 9 pp 691ndash699 2001

[3] R Dammann T Takahashi and G P Pfeifer ldquoThe CpG islandof the novel tumor suppressor gene RASSF1A is intensely


methylated in primary small cell lung carcinomasrdquo Oncogenevol 20 no 27 pp 3563ndash3567 2001

[4] M D Vos C A Ellis A Bell M J Birrer and G J ClarkldquoRas uses the novel tumor suppressor RASSF1 as an effector tomediate apoptosisrdquoThe Journal of Biological Chemistry vol 275no 46 pp 35669ndash35672 2000

[5] R Dammann C Li J H Yoon P L Chin S Bates and G PPfeifer ldquoEpigenetic inactivation of a RAS association domainfamily protein from the lung tumor suppressor locus 3p21 3rdquoNature Genetics vol 25 no 3 pp 315ndash319 2000

[6] M E Reeves S W Baldwin M L Baldwin et al ldquoRas-association domain family 1C protein promotes breast cancercell migration and attenuates apoptosisrdquo BMC Cancer vol 10article 562 2010

[7] Y G Amaar M G Minera L K Hatran D D Strong SMohan and M E Reeves ldquoRas association domain family1C protein stimulates human lung cancer cell proliferationrdquoAmerican Journal of PhysiologymdashLung Cellular and MolecularPhysiology vol 291 no 6 pp L1185ndashL1190 2006

[8] M E Reeves M L Baldwin R Aragon et al ldquoRASSF1Cmodulates the expression of a stem cell renewal gene PIWIL1rdquoBMC Research Notes vol 5 article 239 2012

[9] M E Reeves R J AragonMAlfakhouri et al ldquoRas-associationdomain family 1C protein enhances ereast tumor growth invivordquo Cancer Growth and Metastasis vol 5 pp 27ndash33 2012

[10] E Estrabaud I Lassot G Blot et al ldquoRASSF1C an isoform ofthe tumor suppressor RASSF1A promotes the accumulation of120573-catenin by interacting with 120573TrCPrdquo Cancer Research vol 67no 3 pp 1054ndash1061 2007

[11] G Pelosi C Fumagalli M Trubia et al ldquoDual role of RASSF1 asa tumor suppressor and an oncogene in neuroendocrine tumorsof the lungrdquo Anticancer Research vol 30 no 10 pp 4269ndash42812010

[12] G Malpeli E Amato M Dandrea et al ldquoMethylation-associated down-regulation of RASSF1A and up-regulation ofRASSF1C in pancreatic endocrine tumorsrdquo BMC Cancer vol11 article 351 2011

[13] C J Foley H Freedman S L Choo et al ldquoDynamics ofRASSF1AMOAP-1 associationwith death receptorsrdquoMolecularand Cellular Biology vol 28 no 14 pp 4520ndash4535 2008

[14] M D Vos A Dallol K Eckfeld et al ldquoThe RASSF1A tumorsuppressor activates bax via MOAP-1rdquoThe Journal of BiologicalChemistry vol 281 no 8 pp 4557ndash4563 2006

[15] C Guo X Zhang and G P Pfeifer ldquoThe tumor suppres-sor RASSF1A prevents dephosphorylation of the mammalianSTE20-like kinases MST1 and MST2rdquo The Journal of BiologicalChemistry vol 286 no 8 pp 6253ndash6261 2011

[16] J Avruch M Praskova S Ortiz-Vega M Liu and X F ZhangldquoNore1 and RASSF1 regulation of cell proliferation and of theMST12 kinasesrdquoMethods in Enzymology vol 407 pp 290ndash3102005

[17] H J Oh K-K Lee S J Song et al ldquoRole of the tumor suppres-sor RASSF1A inMst1-mediated apoptosisrdquoCancer Research vol66 no 5 pp 2562ndash2569 2006

[18] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[19] M Yi J Yang X Chen et al ldquoRASSF1A suppresses melanomadevelopment by modulating apoptosis and cell-cycle progres-sionrdquo Journal of Cellular Physiology vol 226 no 9 pp 2360ndash2369 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


2 Materials and Methods

21 Cell Culture The human breast cancer cell line T47Dand lung cancer cell line NCI-H1299 stably overexpressingHA-RASSF1A or HA-RASSF1C were developed as previouslydescribed [6] Cells overexpressing HA-RASSF1A (T47D-1A H1299-1A) HA-RASSF1C (T47D-1C H1299-1C) or theempty MLV backbone (T47D-BB H1299-BB) were grownin RPMI-1640 medium supplemented with 10 calf bovineserum For growth of T47D cells mediumwas supplementedwith 02UmL insulin

22 Western Blot Analysis Western blot analysis was carriedout using standard procedures with detection on the OdysseyInfrared System (LI-COR Biosciences Lincoln NE) Rabbitpolyclonal anti-MST12 and anti-pMST12 (Cell Signaling)HA-tag antibody (Covance) anti-YAP and anti-pYAP (CellSignaling) and goat anti-mouse and donkey anti-rabbitfluorescently labeled secondary antibodies (IRDye 680 926-32220 LI-COR Biosciences) were used

23 Treatment of Cells with TNF-120572 Cells were plated in 96-well plates at 5000 cellswell and were treated the next daywith 1 120583gmL Actinomycin D for 2 hr Cells were then treatedwith 40 120583M TNF-120572 in DMSO (Sigma St Louis MO) for6 hr and then analyzed by western blot Alamar Blue cellviability assays were performed 18 hr after TNF-120572 treatmentas previously described [7] Cell proliferation was measuredby the Alamar Blue assay as previously described [6 7]Data are presented as mean values plusmn SEM and analyzed withStudentrsquos 119905-test Values le005 were considered significant

24 Caspase 37 Activity Assay Cells treated with TNF-120572 for6 hr were assayed for caspase 37 activity using the Apo37caspase activity assay (Promega Madison WI)

25 RNA Isolation and RT-PCR Analysis Tumor andmatched normal tissues were obtained from the WesternDivision of the Cooperative Human Tissue Bank (VanderbiltUniversity TN)

Tumor and normal breast and lung tissues were groundto a powder using a mortar and pestle and the tissuepowder was used to isolate total RNA using PureLink RNAMini Kit (Invitrogen Carlsbad CA) 1120583g of total RNAwas used in reverse transcriptase (RT) reactions using thesuperscript kit (Qiagen Germantown MD) and the RTreactions were subsequently used to set up real-time PCR(RT-PCR) reactions using 1 120583L of RT as a template The RT-PCR reactions were set up using SYBR green PCR mastermix (BioRad Hercules CA) and the PCR reactions wererun using the Opticon 2 PCRmachine (BioRad) Cyclophilinwas used as a loading control Expression of RASSF1A andRASSF1C was assessed by qRT-PCR using RASSF1A andRASSF1C specific primersThe PCR reactions were run usingthe following protocol (1) incubate at 95∘C for 10min (2)incubate at 95∘C for 15 sec (3) incubate for 30 sec (4) go toline 2 for 39 more cycles (5) melt curve from 60∘C to 95∘Cread every 10∘C and (6) incubate at 10∘C forever qRT-PCR

T47D

-BB

T47D

-1A

H12

99-B

B

H12

99-1

A

T47D

-BB

T47D

-1C

H12

99-B

B

H12

99-1

C

HA-1AActin Actin

HA-1C

Figure 1 Breast T47D and lung NCI-H1299 cancer cells sta-bly transduced with MLV backbone or MLV vectors contain-ing HA-RASSF1A or HA-RASSF1C overexpress the transducedprotein as judged by western blots using HA-antibody T47D-BB=T47D transducedwithMLVbackbone T47D-1A=T47D over-expressing HA-RASSF1A T47D-1C=T47D overexpressing HA-RASSF1CH1299-BB=NCI-H1299 transducedwithMLVbackboneand H1299-1A=NCI-H1299 overexpressing HA-RASSF1A H1299-1C =NCI-H1299 overexpressing HA-RASSF1C

reactions were carried out in triplicate and the fold changewas calculated using the 2minusΔΔCT method [18] The RASSF1Aforward primer is 51015840 GGCGTCGTGCGCAAAGGCC 31015840the RASFF1C forward primer is 51015840 CTGCAGCCAAGAG-GACTCGG 31015840 and the reverse primer for both RASSF1Aand RASSF1C is 51015840 GGGTGGCTTCTTGCTGGAGGG 31015840The cyclophilin (housekeeping gene) forward primer is51015840 GCATACAGGTCCTGGCATCT 31015840 and the cyclophilinreverse primer is 51015840 TCTTGCTGGTCTTGCCATTC 31015840 PCRreactions were set in triplicates and cyclophilin was used asan internal loading control and was used to normalize therelative expression levels using the 2minusΔΔCT method [18]

3 Results

31 RASSF1C Reduces the Effects of TNF-120572 on Cell Prolifera-tion TNF-120572 induces cell apoptosis and it has been demon-strated that RASSF1A mediates the apoptotic effects of TNF-120572 In contrast we have previously shown that overexpressionof RASSF1C attenuates apoptosis in breast and lung cancercells In this study we compared the effects of RASSF1Aand RASSF1C overexpression on breast and lung cancer cellproliferation in the presence and absence of TNF-120572 (Figures1 2 and 3) We found that while RASSF1A reduced cell pro-liferation and further enhanced the antiproliferative effectsof TNF-120572 RASSF1C significantly increased cell proliferationand reduced the antiproliferative effects of TNF-120572 on breastand lung cancer cells Our findings suggest that RASSF1A andRASSF1C have antagonistic effects on cell proliferation

32 RASSF1C Reduces Caspase 37 Activation in Breast andLung Cancer Cells Treated with TNF-120572 To further investigatethe antagonism of the antiproliferative effects of TNF-120572 byRASSF1C we assessed the activation of caspase 37 in breastand lung cancer cells overexpressing RASSF1A and RASSF1Cthat were treated with TNF-120572 Both breast and lung cancercells overexpressing RASSF1C showed a significant reductionin caspase 37 activation while RASSF1A overexpressionenhanced caspase 37 activity (Figure 4) This suggests thatRASSF1C has antiapoptotic effects on breast and lung cancercells and further supports antagonistic effects of RASSF1Aand RASSF1C on apoptosis


12500

13000

13500

14000

14500

15000

15500

16000

16500


Abso

rban

ce (a

u)

lowast

(a)

Abso

rban

ce (a

u)

1000

2000

3000

4000

5000

6000

H1299-BB H1299-1A H1299-1C

lowast

(b)


0150030004500600075009000

105001200013500150001650018000


lowast

Abso

rban

ce (a

u)

TNF-120572

(a)

minus + + +

lowast

Abso

rban

ce (a

u)

TNF-1205722000

4000

6000

8000

10000

H1299-BB H1299-BB H1299-1A H1299-1C

(b)


0100000200000300000400000500000600000700000800000900000


lowast

lowast

Casp

ase 3

7 ac

tivat

ion

assa

y

(a)

0

500000

1000000

1500000

2000000

2500000

3000000

lowast

lowast

Casp

ase 3

7 ac

tivat

ion

assa

y

H1299-BB H1299-1A H1299-1C

(b)



0

02

04

06

08

1

12

14

002040608

112141618

TNF-120572

TNF-120572 TNF-120572

H1299-1C

pMST

Actin

pMST

Actin

H1299-BB H1299-1A


+

minus minus



Qua

ntifi

ed p

MST

pro

tein

leve

ls

Qua

ntifi

ed p

MST

pro

tein

leve

ls

T47D-BB

T47D-BB


T47D-1A

T47D-1C

T47D-1C

(a)

0

05

1

15

2

25

3

35

0

1

2

3

4

5

6


Qua

ntifi

ed M

ST p

rote

in le

vels

Qua

ntifi

ed M

ST p

rote

in le

vels

H1299-BB H1299-1C H1299-1A

MST

Actin

MST

Actin

T47D

-BB

T47D

-1C

T47D

-1A

H12

99-B

B

H12

99-1

C

H12

99I-

1A

(b)





0

05

1

15

2

25

3

35

0

1

2

3

4

5

6



Qua

ntifi

ed p

YAP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

TNF-120572pYAP

YAP

Actin

T47D

-BB

T47D

-BB

T47D

-1A

T47D

-1A

T47D

-1C

T47D

-1C

(a)

0

02

04

06

08

1

12

14

16

005

115

225

335

445

Qua

ntifi

ed Y

AP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

TNF-120572pYAP

YAP

Actin

H12

99-B

B

H12

99-B

B

H12

99-1

A

H12

99-1

A

H12

99-1

C

H12

99-1

C

(b)






0

2

4

6

8

10

12

14

1

1A1C

2 3

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(a)

012345678

1 2 3

1A1C

4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(b)



012

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1A N1A T

(a)

minus2

minus1

0

1

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1C N1C T

(b)





4 Discussion



minus6

minus5

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive R

ASS

F1A

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

1A N1A T

(a)

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1C N1C T

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

(b)






0123456789

10


1C1

A m

RNA

ratio

in tu

mor

s

(a)

005

115

225

335

445

5


1C1

A m

RNA

ratio

in tu

mor

s

(b)






5 Conclusions




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


12500

13000

13500

14000

14500

15000

15500

16000

16500


Abso

rban

ce (a

u)

lowast

(a)

Abso

rban

ce (a

u)

1000

2000

3000

4000

5000

6000

H1299-BB H1299-1A H1299-1C

lowast

(b)


0150030004500600075009000

105001200013500150001650018000


lowast

Abso

rban

ce (a

u)

TNF-120572

(a)

minus + + +

lowast

Abso

rban

ce (a

u)

TNF-1205722000

4000

6000

8000

10000

H1299-BB H1299-BB H1299-1A H1299-1C

(b)


0100000200000300000400000500000600000700000800000900000


lowast

lowast

Casp

ase 3

7 ac

tivat

ion

assa

y

(a)

0

500000

1000000

1500000

2000000

2500000

3000000

lowast

lowast

Casp

ase 3

7 ac

tivat

ion

assa

y

H1299-BB H1299-1A H1299-1C

(b)



0

02

04

06

08

1

12

14

002040608

112141618

TNF-120572

TNF-120572 TNF-120572

H1299-1C

pMST

Actin

pMST

Actin

H1299-BB H1299-1A


+

minus minus



Qua

ntifi

ed p

MST

pro

tein

leve

ls

Qua

ntifi

ed p

MST

pro

tein

leve

ls

T47D-BB

T47D-BB


T47D-1A

T47D-1C

T47D-1C

(a)

0

05

1

15

2

25

3

35

0

1

2

3

4

5

6


Qua

ntifi

ed M

ST p

rote

in le

vels

Qua

ntifi

ed M

ST p

rote

in le

vels

H1299-BB H1299-1C H1299-1A

MST

Actin

MST

Actin

T47D

-BB

T47D

-1C

T47D

-1A

H12

99-B

B

H12

99-1

C

H12

99I-

1A

(b)





0

05

1

15

2

25

3

35

0

1

2

3

4

5

6



Qua

ntifi

ed p

YAP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

TNF-120572pYAP

YAP

Actin

T47D

-BB

T47D

-BB

T47D

-1A

T47D

-1A

T47D

-1C

T47D

-1C

(a)

0

02

04

06

08

1

12

14

16

005

115

225

335

445

Qua

ntifi

ed Y

AP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

TNF-120572pYAP

YAP

Actin

H12

99-B

B

H12

99-B

B

H12

99-1

A

H12

99-1

A

H12

99-1

C

H12

99-1

C

(b)






0

2

4

6

8

10

12

14

1

1A1C

2 3

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(a)

012345678

1 2 3

1A1C

4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(b)



012

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1A N1A T

(a)

minus2

minus1

0

1

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1C N1C T

(b)





4 Discussion



minus6

minus5

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive R

ASS

F1A

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

1A N1A T

(a)

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1C N1C T

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

(b)






0123456789

10


1C1

A m

RNA

ratio

in tu

mor

s

(a)

005

115

225

335

445

5


1C1

A m

RNA

ratio

in tu

mor

s

(b)






5 Conclusions




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

02

04

06

08

1

12

14

002040608

112141618

TNF-120572

TNF-120572 TNF-120572

H1299-1C

pMST

Actin

pMST

Actin

H1299-BB H1299-1A


+

minus minus



Qua

ntifi

ed p

MST

pro

tein

leve

ls

Qua

ntifi

ed p

MST

pro

tein

leve

ls

T47D-BB

T47D-BB


T47D-1A

T47D-1C

T47D-1C

(a)

0

05

1

15

2

25

3

35

0

1

2

3

4

5

6


Qua

ntifi

ed M

ST p

rote

in le

vels

Qua

ntifi

ed M

ST p

rote

in le

vels

H1299-BB H1299-1C H1299-1A

MST

Actin

MST

Actin

T47D

-BB

T47D

-1C

T47D

-1A

H12

99-B

B

H12

99-1

C

H12

99I-

1A

(b)





0

05

1

15

2

25

3

35

0

1

2

3

4

5

6



Qua

ntifi

ed p

YAP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

TNF-120572pYAP

YAP

Actin

T47D

-BB

T47D

-BB

T47D

-1A

T47D

-1A

T47D

-1C

T47D

-1C

(a)

0

02

04

06

08

1

12

14

16

005

115

225

335

445

Qua

ntifi

ed Y

AP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

TNF-120572pYAP

YAP

Actin

H12

99-B

B

H12

99-B

B

H12

99-1

A

H12

99-1

A

H12

99-1

C

H12

99-1

C

(b)






0

2

4

6

8

10

12

14

1

1A1C

2 3

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(a)

012345678

1 2 3

1A1C

4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(b)



012

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1A N1A T

(a)

minus2

minus1

0

1

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1C N1C T

(b)





4 Discussion



minus6

minus5

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive R

ASS

F1A

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

1A N1A T

(a)

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1C N1C T

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

(b)






0123456789

10


1C1

A m

RNA

ratio

in tu

mor

s

(a)

005

115

225

335

445

5


1C1

A m

RNA

ratio

in tu

mor

s

(b)






5 Conclusions




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

05

1

15

2

25

3

35

0

1

2

3

4

5

6



Qua

ntifi

ed p

YAP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

TNF-120572pYAP

YAP

Actin

T47D

-BB

T47D

-BB

T47D

-1A

T47D

-1A

T47D

-1C

T47D

-1C

(a)

0

02

04

06

08

1

12

14

16

005

115

225

335

445

Qua

ntifi

ed Y

AP

prot

ein

leve

lsQ

uant

ified

pYA

P pr

otei

n le

vels




TNF-120572

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

H1299-BB H1299-BB H1299-1A H1299-1A H1299-1C H1299-1C

TNF-120572pYAP

YAP

Actin

H12

99-B

B

H12

99-B

B

H12

99-1

A

H12

99-1

A

H12

99-1

C

H12

99-1

C

(b)






0

2

4

6

8

10

12

14

1

1A1C

2 3

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(a)

012345678

1 2 3

1A1C

4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(b)



012

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1A N1A T

(a)

minus2

minus1

0

1

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1C N1C T

(b)





4 Discussion



minus6

minus5

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive R

ASS

F1A

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

1A N1A T

(a)

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1C N1C T

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

(b)






0123456789

10


1C1

A m

RNA

ratio

in tu

mor

s

(a)

005

115

225

335

445

5


1C1

A m

RNA

ratio

in tu

mor

s

(b)






5 Conclusions




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

2

4

6

8

10

12

14

1

1A1C

2 3

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(a)

012345678

1 2 3

1A1C

4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive m

RNA

expr

essio

n in

nor

mal

tiss

ue

(b)



012

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1A N1A T

(a)

minus2

minus1

0

1

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

1C N1C T

(b)





4 Discussion



minus6

minus5

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive R

ASS

F1A

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

1A N1A T

(a)

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1C N1C T

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

(b)






0123456789

10


1C1

A m

RNA

ratio

in tu

mor

s

(a)

005

115

225

335

445

5


1C1

A m

RNA

ratio

in tu

mor

s

(b)






5 Conclusions




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


minus6

minus5

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Relat

ive R

ASS

F1A

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

1A N1A T

(a)

minus4

minus3

minus2

minus1

0

1

2

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1C N1C T

Relat

ive R

ASS

F1C

mRN

A ex

pres

sion

in tu

mor

ver

sus n

orm

al ti

ssue

s

(b)






0123456789

10


1C1

A m

RNA

ratio

in tu

mor

s

(a)

005

115

225

335

445

5


1C1

A m

RNA

ratio

in tu

mor

s

(b)






5 Conclusions




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0123456789

10


1C1

A m

RNA

ratio

in tu

mor

s

(a)

005

115

225

335

445

5


1C1

A m

RNA

ratio

in tu

mor

s

(b)






5 Conclusions




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article the rassf1 gene and the opposing effects

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