Thesis

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Abstract

The cell cycle is a highly controlled mechanism composed of G1, S, G2, and M phases,

with the transitions between these phases regulated in part by cyclin dependent kinases

(CDKs). CDK inhibitors (CKIs) including p21 and p27 are key proteins that arrest the cell in G1

to prevent division when the cell is under stress or possesses DNA damage. Additionally, p27

expression is upregulated in quiescence (G0), when the cell is not actively cycling. p27 is

targeted for degradation at the G0/G1 transition, and again at the G1/S transition when p21 is

also degraded, though the order of cell cycle protein degradation at G1/S, and its impact on

normal cell proliferation, is unknown. The purpose of this study is to develop a fusion protein

biosensor that mirrors endogenous p27 expression in mammalian cells. We generated two

constructs containing the p27 gene fused C-terminally with Yellow Fluorescent Protein (Venus

(Ven)). We transfected cells with one such construct and compared p27-Ven levels by

performing time course synchronizations and serum starvation experiments visualized via

immunoblot. Contrary to our expectations, p27-Ven was not degraded at the G1/S transition, nor

were levels upregulated in G0. We therefore cannot currently consider our fusion protein a

reliable biosensor. Interestingly, in other studies, functional N-terminally tagged p27 biosensors

have been developed. We thus produced Ven-p27 constructs and will investigate them as

potential alternatives. Once we develop an effective biosensor, we will produce a

high-resolution temporal map of protein degradation primarily via single cell analysis using live cell

imaging. By perturbing CKI and other cell cycle protein degradation with drugs and siRNAs, we

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Introduction

Cells proliferate by progressing through a series of phases known collectively as the cell

cycle. The transitions between the phases are tightly controlled by cell cycle checkpoints, which

are periods when the cell is kept from dividing until certain intracellular and extracellular

conditions are met. Many protein classes, such as cyclins and cyclin-dependent kinases (CDKs),

form crucial complexes that provide signals to downstream effectors, which permit the transition

to the next phase of the cycle and induce events such as centrosome duplication and DNA

replication via protein phosphorylation (Murray, 2004). The ability of cyclin-CDK complexes to

promote cellular growth is directly regulated by the levels of CDK inhibitor proteins (CKIs).

CKIs prevent cell cycle progression beyond a particular checkpoint by binding to cyclin-CDK

complexes and blocking their enzymatic activity (Coqueret, 2003). In this way, CKIs serve a

critical function as tumor suppressors and control the rate of proliferation and responsiveness of

cell cycle progression to extracellular factors such as substrate adhesion and growth factors (He,

Wang, Chen, & Guan, 2012). A significant majority of human cancers (90%) have an altered

expression of certain CKIs, CDKs, and cyclins within the transformed cells; this altered

expression causes cell proliferation to progress rapidly and without regulation (Bonelli, Tuccillo,

Borrelli, Schiattarella, & Buonaguro, 2014).

The CKI p27 has demonstrated an important role in both cell differentiation and

proliferation, with increased levels of p27 seen following differentiation signals and decreased

levels seen following growth factor signaling (J. Slingerland & Pagano, 2000). The protein p27

inhibits cyclin E/CDK2 and cyclin A/CDK2 complexes, leading to cellular arrest at G1 (Lee &

Kim, 2009). After p27 degradation occurs, these complexes are responsible for S phase onset

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end of G1 (J. M. Slingerland et al., 1994), though p27 is not degraded entirely at this point in the

cell cycle. It is known that the protein exists at reduced levels throughout the cell cycle in the

majority of proliferating cells in cell lines such as BALB/c 3T3 (Agrawal, Dong, Wang, Kayda,

& Pledger, 1995). High expression of p27 is seen in cells that are in G0 (quiescence), and

degradation of p27 occurs in cells that re-enter the cell cycle at G1 (Oki et al., 2014).

Although the timing and order of fluctuations in p27 expression relative to other proteins

at high temporal resolution is unknown, the mechanisms by which p27 accumulates and degrades

are well understood. A major regulator of p27 expression is the ubiquitin proteasome system,

which is responsible for ubiquitinating 80-90% of intracellular proteins (Shen, Schmitt, Buac, &

Ping Dou, 2013). Three types of enzymes mediate this process—E1, E2, and E3—and serve to

activate, conjugate, and ligate ubiquitin, respectively, in a sequence of events that results in a

protein substrate attached to a poly-ubiquitin chain. Once ubiquitinated, proteins are guided to

the 26S proteasome complex for removal of ubiquitin chains before protein degradation occurs.

The E3 enzyme in particular becomes part of the ligase complex SCFSKP2, which facilitates p27 degradation (Shen et al., 2013). Another major component of this complex is S-phase

kinase-associated protein 2 (SKP2) and is a well-known protooncogene and F-box protein. SKP2 plays

a role in mediating p27 degradation at the G1/S transition (Bochis, Irimie, Pichler, & Neagoe,

2015). Further, p27 degradation is regulated by SKP2 at the S/G2 transition, as p27 accumulates

at this transition in mice lacking the Skp2 gene (Nakayama et al., 2004). Unlike during the G1/S

and S/G2 transitions, SKP2 is not present in the cell to degrade p27 at the G0/G1 transition, yet

p27 is nevertheless degraded (Hara et al., 2001). Explaining this discrepancy, a pathway has

been discovered linking p27 degradation to Kip1 ubiquitylation-promoting complex

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exported from the nucleus (Kotoshiba, Kamura, Hara, Ishida, & Nakayama, 2005). Nuclear

export and subsequent cytoplasmic localization occurs due to phosphorylation of the Serine 10

residue of p27. However, cytoplasmic localization does not appear to be necessary for p27

proteolysis as was once thought (Rodier et al., 2001).

A large amount of research has been done regarding proteins present in the S and M

phases of the cell cycle, but the cell cycle transitions have gone relatively unexplored, leaving

the exact accumulation and degradation of cell cycle proteins at critical transitions unknown. In

this study, we seek to address this void, focusing on the CKI p27. We generated two plasmid

DNA constructs containing the p27 gene (CDKN1B) C-terminally tagged to Yellow Fluorescent

Protein (Venus (Ven)). Fluorescent tagging is an appropriate method to use for obtaining

information regarding protein accumulation and degradation timing, as we are able to easily

visualize the fluctuations in fluorescence via a number of microscopy techniques and eventually

live cell imaging. After suspecting that the p27-Ven fusion protein was not a reliable biosensor

to mirror endogenous p27 expression, and with the knowledge that others in our field have also

struggled with C-terminally tagged fusion proteins (Oki et al., 2014), we generated two

N-terminally tagged Ven-p27 constructs that will undergo rigorous testing to verify if they are more

effective and dependable biosensors.

Once we determine which fusion proteins, if any, are effective biosensors, we will create

a high-resolution temporal map of protein degradation order at the G1/S and S/G2 transitions

using these as well as other constructs coding for cell cycle proteins such as p21 and

Cdc6. Future experiments will involve altering the level and order of protein expression and

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degradation impacts cell growth. This knowledge will be useful in further investigation of

diseases such as cancer that are driven by deviation from normal cellular proliferation control.

Materials and Methods

Plasmid Preparation

We generated multiple C-terminally and N-terminally tagged fluorescent plasmid

constructs (p27-Ven and Ven-p27, respectively) via two reaction cloning protocols: Gateway®

BP and LR cloning (Thermo Scientific, DE) and single-step isothermal DNA recombination

(Gibson et al., 2009). Using PCR and Q5® high fidelity DNA polymerase (NEB, MA), we

amplified the CDKN1B gene sequence in the presence of forward and reverse primers specific to

each construct design (see Appendix). Electrophoretic gels used to separate DNA for subsequent

purification steps or verification of cloning success were composed of 1% agarose. We

conducted gel extraction and column purification steps using the appropriate QIAQuick® DNA

purification protocols (QIAGEN, MD). For Gateway® cloning, we subcloned the DNA

fragments into the donor vector pDONR221 using the Gateway® BP reaction protocol and used

electrocompetent DH5αE. coli cells in transformation.

After performing the BP reaction and transformation, we plated on kanamycin plates and

incubated overnight. We performed PCR on colonies using standard techniques to screen for

successful transformations. We then amplified and isolated the plasmid via a QIAQuick®

mini-prep protocol (QIAGEN, MD). At this point, we subcloned the entry vectors into destination

vectors (see Appendix) using the standard Gateway® LR reaction protocol and transformed into

DH5αE. coli. pLenti PGK Neo DEST (w531-1) and pLenti PGK Puro DEST (w529-2) were

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2009). pcDNA5/FRT/TO-Venus-Flag-Gateway (1124) was a gift from Jonathon Pines

(Addgene plasmid # 40999). The vectors will henceforth be referred to as the following:

Destination Vector Name New Vector Name Associated Expression _{Vector Name}

pcDNA5/FRT/TO-Venus-Flag-Gateway (1124) pDESTJC13 pEXPJC13-p27-Ven

pLenti PGK Puro DEST

(w529-2) pLenti-puro pLenti-puro-Ven-p27

pLenti PGK Neo DEST

(w531-1) pLenti-neo pLenti-neo-Ven-p27

We employed the same screening methods after completing the LR protocol though

selected with ampicillin rather than kanamycin. After construct screening and purification, we

confirmed cloning success through DNA sequencing via UNC sequencing facilities (UNC-CH,

NC). We generated all constructs except pEXPJC17-p27-Ven using the above method. For

pEXPJC17-p27-Ven, a nourseothricin derivative of pEXPJC13-p27-Ven, we subcloned the

nourseothricin resistance gene into pEXPJC16-p27-Ven (another derivative of

pEXPJC13-p27-Ven) after removing its puromycin resistance gene using a single-reaction isothermal cloning

method (Gibson et al., 2009). We employed the same general methods for purification,

transformation, and verification as with the Gateway® cloning scheme.

Cell Cultures and Stable Cell Line Generation

We maintained all cell lines in Dulbecco's modified Eagle's medium (DMEM) (Sigma) +

10% fetal calf serum (FBS), L-Glutamine, and penicillin/streptomycin. To generate our stable

cell lines, we transfected pEXPJC13-p27-Ven into human embryonic kidney HEK 293T

mammalian cells obtained from American Type Cell Collection using a standard transfection

protocol with polyethyleneimine (PEI). After transfection and fluorescence confirmation, we

developed pEXPJC17-p27-Ven and went forward with usage of that construct for its preferred

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U2OS-TRex cells, which were a gift from John Aster, for further experimentation (Malecki et al.,

2006). TRex cells utilize the Flp-In™ T-REx™ System which enables the use of FRT sites

within the plasmid to perform recombinase-mediated DNA recombination and generate stable,

inducible expression cell lines (Thermo Scientific, DE). Our pEXPJC17-p27-Ven construct is

under a doxycycline-inducible system (dox). For the pLenti constructs, we employed a standard

lentiviral transfection protocol to transfect the cells using pVSVG and ΔNRF plasmids. We then

performed a transduction into the human telomerase reverse transcriptase immortalized retinal

pigment epithelial cell line hTERT-RPE1 using a standard protocol with 8 µg/mL polybrene.

The expression of the Ven-p27 fusion protein generated in these cells is not

doxycycline-inducible but rather constitutively expressed.

Microscopy

We imaged all cells using a Carl Zeiss Axiovert 40 CFL inverted microscope with a 10X

objective and 10X lens. We visualized fluorescence using an X-Cite® 120Q wide-field

fluorescence microscope excitation light source. We utilized ImageJ software to alter DIC

images to grayscale and fluorescence images to yellow from their original green for improved

viewing and better understanding of our results. Because we did not quantify our images, we

also used ImageJ to enhance contrast and decrease background noise so that dimly fluorescing

cells could be better observed for qualitative analysis.

Protein Lysate Preparation

We prepared whole cell lysates by washing and collecting cells in trypsin and then

centrifuging the cells at 2,000 rpm for 2 minutes. After removing the trypsin via pipetting, we

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this process once and then resuspended the pellet in 2X Laemmli sample buffer (SDS) + 10% β

-mercaptoethanol (BME). We then boiled the lysates for 5 minutes before immunoblotting.

Immunoblot Analysis

For all of our immunoblots, we used 10% polyacrylamide gels and loaded 2.5 µL of

Precision Plus Protein™ Dual Color Standards ladder (BioRad, CA) and 15-20 µL of protein

lysate. We electrophoresed the gels at 170V for 70 minutes and transferred at either 110V for 75

minutes or 20V overnight. We transferred all blots to PVDF (Thermo Scientific, DE) except for

the serum starvation and fusion protein comparison blots, for which we used nitrocellulose

membranes (GE Healthcare Life Sciences, UK). Before blocking, we added Ponceau S solution

to confirm uniform protein loading concentration (Sigma). We blocked for 1-2 hours in 5%

nonfat dry milk/Tris buffered saline with Tween® (TBST).

After blocking, we added primary antibodies in 2.5% milk/TBST for 1 hour then washed

with TBST 3 times for 5 minutes each. When probing for p27, we used rabbit polyclonal p27

primary antibody (C-19, Santa Cruz Biotechnology, sc-528) at 1:3,000. For our cyclin A

immunoblot, we probed with cyclin A polyclonal primary antibody (C-19, Santa Cruz

Biotechnology, sc-596) at 1:2,000. We added donkey anti-rabbit HRP secondary antibody

(Jackson ImmunoResearch Laboratories, PA) to all blots at 1:10,000 in 1.25% milk/TBST for 1

hour. We washed all blots in TBST 3 times for 5 minutes each before preparing blots for

visualization using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life

Sciences, UK).

Time Course Synchronizations

We conducted two time course synchronization experiments: a thymidine-nocodazole

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for both of these experiments. In the thymidine-nocodazole block and release experiment, we

blocked cells in thymidine to synchronize at the beginning of S phase. Upon release, we allowed

the cells to progress through one cell cycle before blocking the cells with nocodazole, which

subsequently synchronized the cells at the G2/M phase transition. In the double thymidine block

and release experiment, we synchronized cells at the G1/S transition for lysate collection,

enabling us to visualize p27-Ven and endogenous p27 expression in S phase rather than G1.

For our thymidine-nocodazole block and release experiment, we plated cells at 25%

confluence in 60 mm dishes for 3 doxycycline conditions (0, 5, and 20 µg/mL). After 24 hours

had elapsed, we treated the cells with 2.5 mM thymidine for 24 hours. We then washed the cells

twice with 1X PBS and added DMEM + 10% FBS + 100 ng/mL nocodazole + doxycycline for

16 hours. At this point, we collected each well’s media into one of three 50 mL conicals based

on doxycycline concentration and centrifuged at 2,000 rpm for 5 minutes. We aspirated the

liquid and resuspended each conical in DMEM + 10% FBS. Before replating the liquid, we

washed the cells twice with 1.5 mL 1X PBS. We collected each time point by trypsinization

following our protein lysate protocol for a total of nine time points.

For our double thymidine block and release experiment, we plated cells at roughly 20%

confluence in 6-well dishes with multiple doxycycline conditions. One day after plating, we

blocked cells in 2.5 mM thymidine for 18 hours. We released the cells by washing twice with

1X PBS and replaced the media with DMEM + 10% FBS. After 8 hours, we blocked again in

2.5 mM thymidine and added doxycycline for 18 hours before releasing the cells using the same

protocol. We collected two time points following our protein lysate protocol.

Serum Starvation

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JC17-p27-Ven, RPE-TRex as a control for p27 expression in normal cells (a gift from the

Jackson lab), and U2OS-TRex-JC13-GFP as a control for cells containing an expression plasmid.

We plated cells into 6-well dishes in 2 mL DMEM + 10% FBS and had multiple conditions per

cell line (Figure A1). The day after we plated the cells, we removed the medium and replaced

with either DMEM + 10% FBS or 0% FBS following our conditions. After 24 hours of

starvation, we added 10 µg/mL doxycycline to the appropriate p27-Ven wells to induce fusion

expression. Before collecting each well as a protein lysate, we visualized the cells using

differential interference contrast (DIC) microscopy and a YFP filter.

Results

! The primary goal of this study is to develop and verify fusion protein biosensors that

mirror endogenous protein expression. Our overall expression strategy is to develop plasmid

DNA constructs that contain the genetic sequence for our desired fusion protein, p27-Ven. The

fusion protein sequence bears a promoter region that is doxycycline-inducible. In utilizing as

doxycycline-inducible system, we can determine an appropriate doxycycline concentration to use

going forward in which we can induce the same level of fusion protein p27 expression as

endogenous p27 expression. We elected to use a U2OS cell line because we are particularly

interested in protein dynamics within transformed cells and have previously used U2OS cells

when developing other cell lines we hope to compare to our p27-Ven lines.

pEXPJC13-p27-Ven expresses in HEK 293T cells

To develop an effective fusion protein biosensor, we first generated a C-terminally

tagged doxycycline-induced plasmid construct: pEXPJC13-p27-Ven. We then transfected the

plasmid into HEK 293T mammalian cells to confirm our construct’s ability to fluoresce in vivo

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HEK 293T cells fluoresced, indicating successful cloning and construct development (Figure 1,

A and B). p27-Ven did not appear localized in a specific region of the cell due to overexpression

of the protein hindering qualitative analysis of the composite image (Figure 1C). The result of

the transfection enabled us to go forward in transducing U2OS cells with this construct.

pEXPJC17-p27-Ven expresses in U2OS cells

After confirming that our p27-Ven fusion protein was capable of producing visible

fluorescence, we designed and constructed a plasmid with the more effective drug resistance of

nourseothricin rather than hygromycin. By utilizing a stronger drug resistance marker, we

decreased the length of time required for selection to generate stable cell lines, which provided

us with the ability to generate multiple cell lines that bear this construct more efficiently than

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would have been possible using a hygromycin resistance system. We designed and produced the

plasmid construct pEXPJC17-p27-Ven to accomplish this objective. Once the construct was

generated, we performed a transfection using HEK 293T cells before transducing U2OS

cells. We confirmed that the transduction was successful and that pEXPJC17-p27-Ven exhibited

fluorescence via microscopy with a YFP filter after inducing expression with doxycycline

(Figure 2B). Fluorescence levels appeared variable across individual cells that exhibited

fluorescence, though a sizeable proportion of the cells were fluorescing (Figure 2, A and B). The

fluorescence appeared localized to the nuclei of the fluorescing cells due to the rounded shape of

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the fluorescence and composite images that were generated (Figure 2, C and D). From these

data, we concluded that U2OS cell machinery was effectively translating the fusion protein and

that the protein was capable of fluorescing as expected. The protein is also localizing in the

same manner as is expected of functioning endogenous p27, though the functionality of the

fusion protein cannot be confirmed to mirror endogenous p27 based on these conclusions.

p27-Ven and endogenous p27 express abnormally in cell synchronizations

Once we generated a stable cell line and established that p27-Ven fluoresces in U2OS

cells, we aimed to verify whether or not the fusion protein exhibited similar dynamics as

endogenous p27. We first performed a thymidine-nocodazole block and release synchronization

to visualize the expression of p27-Ven and endogenous p27 as the cells synchronously moved

through G1 into S phase (Figure 3). Blocking initially in thymidine synchronizes the cells at the

beginning of S phase. After releasing the cells from thymidine and allowing the cells to progress

through one cell cycle from that point, we blocked in nocodazole, which serves to improve

synchronization efficiency by synchronizing the cells at the G2/M phase transition. Upon

releasing the cells from nocodazole, we expected to observe both endogenous p27 and p27-Ven

expression decrease as the cells progressed from G1 into S phase.

In these as well as subsequent blots, a prominent nonspecific band appeared directly

above the fusion protein p27-Ven. We were able to establish which band was the nonspecific

band by comparing a U2OS-GFP cell line to our p27-Ven line and probing for p27. This blot

also confirmed that we had doxycycline-inducible Ven expression (Figure A2). Leaky

p27-Ven expression was observed in the cells that received 0 µg/mL doxycycline, as the fusion

protein should only be induced in the presence of doxycycline (Figure 3A). This leaky

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transcription of the genes encoding p27-Ven in the absence of doxycycline stimulation. Further,

endogenous p27 was not visible on the blot until late in the time course due to the band being

close to the bottom of the gel and transferring off of the membrane during immunoblotting.

Regardless of this error, the endogenous p27 level appeared nearly equivalent to that of the

fusion protein at the same time points (7h, 8h, and 9h). We had expected that endogenous p27

levels would decrease within this timeframe, though any explanation for this discrepancy

between our expected and actual results would be purely speculative until this experiment is

repeated and endogenous p27 expression is fully observed.

Although previous studies have demonstrated that p27 is degraded at the G1/S transition

(J. M. Slingerland et al., 1994), p27-Ven did not appear to be degraded at the G1/S transition in

the leaky 0 µg/mL doxycycline cells (Figure 3A). Similarly, in the cells that received 20 µg/mL

doxycycline, p27-Ven levels remained constant throughout the time course (Figure 3C). It

appeared that p27-Ven expression decreased very slightly in the 5 µg/mL doxycycline cells over

the time course (Figure 3B). However, contrary to our expectations for endogenous p27

expression, expression remained constant in both the 20 µg/mL and 5 µg/mL doxycycline cells,

if not increasing in the 20 µg/mL doxycycline time course (Figure 3, B and C). Possible

explanations for this trend include that the cells did not effectively synchronize or that the fusion

protein was not degraded with the same kinetics in which endogenous p27 was degraded.

We attempted to use cyclin E to confirm when the G1/S transition occurred during the

time course but grossly overestimated the antibody dilution factor, which resulted in an

overprobed blot (image not shown). Nevertheless, the G1/S transition was expected to occur

during this timeframe based on previous studies with U2OS cells (Grant et al., 2013; Laoukili et

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course synchronization three times (twice when using a 6 hour time course and once with a 9

hour time course (Figure 3)). These results caused us to grow concerned that by inducing

p27-Ven expression in the cells, we were potentially impacting the ability of the cells to move into S

phase effectively due to the role p27 plays in cell cycle inhibition at G1 phase.

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Figure 4. Immunoblot of double thymidine time course synchronization. U2OS-JC17-p27-Ven cells were blocked in thymidine for 18 hours, were released for 8 hours, and then blocked again in thymidine for 18 hours before being collected at various time points. Thy 0 cells received no thymidine treatment and were thus an unsynchronized control. Variable doxycycline

concentrations were used (0, 0.5, 1, 3, 5, 10, and 20 µg/mL). Cyclin A was used to confirm that the cells were in S phase for select doxycycline concentrations (0, 5, and 20 µg/mL). Ponceau S served as a loading control.

In an attempt to observe p27-Ven expression in S phase, we performed a double

thymidine block and release experiment. Because thymidine blocks cells at the beginning of S

phase, this experimental design enabled us to look for changes in p27-Ven and endogenous p27

expression at the start and into S phase. We expected to see both p27-Ven and p27 levels remain

at a low level as the cells progressed through S phase, depending on the exact timing of p27

degradation relative to the cell cycle.

Endogenous p27 expression remained constant across time points as well as relatively

constant across doxycycline concentrations, as expected (Figure 4). p27-Ven levels fluctuated

inconsistently; the 0, 1, and 5 µg/mL doxycycline cells (lanes 3 and 4; 7 and 8; and 11 and 12,

respectively) demonstrated a possible increase in p27-Ven, while the unsynchronized 0 µg/mL

doxycycline cells (lanes 1 and 2) demonstrated a decrease in p27-Ven. The remaining cells

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determine if the cells were in S phase, when cyclin A levels are high. Cyclin A levels remained

constant and high across time points and doxycycline concentrations.

Endogenous p27 levels decrease in serum-starved U2OS cells

Because of the inconsistencies regarding the anticipated and actual results of the

expression of p27-Ven in U2OS cells, we suspected that our fusion protein might not be an

effective biosensor for endogenous p27 expression at the G1/S phase transition. Despite this

suspicion, we recognized that we could attempt to employ the biosensor to examine p27

expression as cells transition between G1 and G0 phase. To determine if p27-Ven expression

was comparable to endogenous p27 expression in G0, we performed a serum starvation

experiment (Figure 5). In this experiment, we compared U2OS cells with p27-Ven to U2OS-

cells with GFP, which provided a control for any impact the addition of a fluorescent protein

might have on cells, as well as for any differences across U2OS cell lines. We also compared

our p27-Ven cell lines to an RPE cell line, which provided a positive control for endogenous p27

expression in non-transformed cells. Additionally, we compared wells that were allowed to

become confluent with cells to those that were not allowed to do so, as confluence and contact

inhibition can trigger cells to enter G0 even in the presence of FBS (Hayes et al., 2005; Vinals &

Pouyssegur, 1999). In addition, we examined the effect doxycycline induction had on p27-Ven

expression and compared serum-starved cells to cells receiving FBS. We expected to observe

high p27-Ven and endogenous p27 levels in serum-starved cells as well as confluent cells in

comparison to non-serum-starved cells and non-confluent cells.

After immunoblotting and exposing the blot for a prolonged amount of time, we only

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that were not confluent upon collection (Figure 5A). Because of this, we focused our attention

on determining whether p27 endogenous expression was consistent with our expectations.

Although there was a slight increase in p27 expression in the serum-starved cells that

were neither confluent nor doxycycline-induced, there was a dramatic decrease in p27 expression

in the serum-starved confluent cells within this group when compared with the FBS+ confluent

cells (Figure 5A). This was inconsistent with our expectations. Further, this trend was also seen

in doxycycline-induced cells, which was expected since doxycycline should not impact p27

endogenous expression.

While the p27-Ven cell line demonstrated results in the serum starvation that conflict

with dependable results in other studies related to p27 expression in G0, the endogenous p27 in

the controls used in this experiment behaved as expected. In the U2OS-GFP cells, serum

starvation yielded roughly equivalent expression of endogenous p27 as was seen when we forced

cells to grow to confluence (Figure 5A). The FBS+ cells that were not confluent demonstrated

low levels of endogenous p27. These results were also demonstrated in the RPE cells as

predicted. This experiment was repeated three times with small adjustments in methodology

until the results in this paper were obtained. Each experiment indicated results similar to those

demonstrated.

Prior to collection of the protein lysates used in the serum starvation immunoblot, we

imaged the U2OS-p27-Ven cells under DIC and with a YFP filter to visually characterize the

fluorescence. We opted to complete this step in the event that few cells were expressing greater

amounts of p27-Ven than other cells and thus biasing our immunoblot data. Cells treated with

10 µg/mL doxycycline and supplied FBS had roughly equivalent numbers and intensities of

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doxycycline (Figure 5, B and C, E and F). Variable intensity of fluorescence between cells was

slightly more pronounced in the FBS+ cells than in the serum-starved cells, though variability

was present in both cell populations. p27-Ven localization was nuclear in both cell populations

(Figure 5, D and G). Variable fluorescence intensity yet consistently nuclear localization was

expected results based on previous visualization of this cell line. However, we did not expect to

see a population of cells in the FBS+ cells that were fluorescing much brighter than the brightest

fluorescing cells in the serum-starved population.

Ven-p27 as a reliable marker for endogenous p27 expression

Because of our difficulties verifying the p27-Ven fusion protein as a biosensor, we

reached the decision that this fusion protein is likely not a reliable biosensor around which we

should base future research endeavors. We also discovered that a study by Oki et al. has

produced both C- and N-terminally tagged mutant p27 fusion proteins, with only the N-

terminally tagged fusion protein being declared a successful biosensor (Oki et al., 2014). Thus,

we developed an N-terminally tagged p27 fusion protein, Ven-p27, to have a better chance of

successfully creating a biosensor for endogenous p27 expression (Figure 6A). While the fusion

proteins generated by Oki et al. involve a defective p27K- mutant that does not possess CDK

activity, we designed our fusions using wild-type p27 so that they might function as biosensors

for endogenous p27 expression. In designing a new construct with this scheme, we shifted away

from U2OS cells and focused on generating stable RPE cell lines. We chose to use a

non-transformed cell line in the event that the U2OS cells were not responding as expected because

they were transformed and thus behaved aberrantly. We also moved from using a

doxycycline-inducible system to a lentiviral system to enable faster experimentation. We developed and then

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Although no fluorescence was visualized using microscopy with a YFP filter in the

hTERT-RPE1 transduced cells (image not shown), immunoblot results demonstrated that the

transductions were in fact successful for both the pLenti-neo-Ven-p27 and pLenti-puro-Ven-p27

constructs. Ven-p27 levels were low in these constructs and roughly equivalent to the level of

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fusion protein created in the uninduced JC17-p27-Ven cells (Figure 6B). Control

U2OS-JC17-p27-Ven cells with and without doxycycline both demonstrated fluorescence, with leaky

expression seen in uninduced cells and greater expression in induced cells. Localization of the

fusion protein was nuclear and was consistent with previous microscopy (Figure 6, C-H).

Discussion

In this study, we sought to develop fluorescent p27 plasmid constructs to serve as

biosensors for endogenous p27 expression in mammalian cells. To this end, we generated

C-terminally and N-C-terminally tagged fluorescent p27 constructs and investigated their

functionality as biosensors. We focused on the protein p27 due to its involvement in not only

cell cycle proliferation but also in cancer. It has been shown that p27 has a significant impact on

cell proliferation, with p27 degradation and deregulation being linked to poor prognoses in

multiple forms of cancers (Chiarle et al., 2000). Cancers such as colon, breast, nasopharyngeal,

and others demonstrate lower nuclear p27 levels in more advanced stages of cancer, indicating

an inverse correlation between p27 expression and a desirable prognosis (Alkarain, Jordan, &

Slingerland, 2004; Chu, Hengst, & Slingerland, 2008; He et al., 2012; Liu et al., 2013; J.

Slingerland & Pagano, 2000).

We successfully developed hygromycin resistant pEXPJC13-p27-Ven and transfected it

into HEK 293T cells to validate fusion protein fluorescence. Instead of further utilizing

pEXPJC13-p27-Ven, we modified the construct to obtain a construct with the more favorable

drug resistance of nourseothricin (pEXPJC17-p27-Ven). We confirmed the success of this

cloning and subsequent transduction after visualizing fluorescent U2OS cells.

In studying the kinetics of our fusion protein, we saw that both the fusion protein and

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course experiment. Although we repeated this experiment multiple times in an attempt to obtain

a greater protein yield and confirm visualization of the G1/S transition, we never saw p27-Ven or

endogenous p27 levels fluctuate. Our double thymidine block also indicated our fusion protein

might not be functioning properly. Endogenous and fusion protein p27 levels remained constant

and elevated in this experiment. Additionally, cyclin A levels remained constant, which

demonstrated the cells were in S phase and low p27 levels should have been observed.

There are a number of reasons that may explain these unanticipated results. In both

experiments, the cells may not have synchronized very well. Additionally, in the

thymidine-nocodazole experiment, the cells may have had an abnormally prolonged G1 phase due to the

addition of p27-Ven, which is a cell cycle inhibitor. We also considered the possibility that a

small proportion of cells were overexpressing the fusion protein in any given experiment. This

cell population may be distorting the data because immunoblotting averages protein expression

across a population. Also conceivable is that the U2OS cells do not regulate p27 properly as a

consequence of their transformed status.

In the future, we plan to utilize single cell analysis via live cell imaging to avoid

continually experiencing difficulties when interpreting immunoblot data. We will also repeat

these experiments and include a control cell line such as untransfected U2OS cells to confirm

that the cells are indeed cycling. In this study, our cyclin E blot was very overprobed, leading to

possible misinterpretation of the blot as well as no clear indication that the cells actually

transitioned from G1 to S phase. Due to this error, we will work to successfully probe future

thymidine-nocodazole blots to look for cyclin E or another protein such as p21 that experiences a

change in expression at the G1/S transition. Although we were able to include a nonspecific

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results as formal loading controls. We were unable to include these controls in this study

because we did not image our blots when staining with Ponceau S during experimentation.

Like our time course synchronization experiments, our investigation of p27-Ven and

endogenous p27 expression via serum starvation yielded unexpected results. Interestingly, we

saw minimal p27-Ven expression via immunoblotting yet clearly saw fluorescence in some cells

when imaging the cells using a YFP filter. This disparity may be explained by our previous

suggestion that if few cells were expressing the fusion protein, average fusion protein expression

would appear decreased. However, fluorescence in this experiment generally appeared

consistent with fluorescence seen in past experiments. Further experimentation is necessary

before we can identify and understand the cause of this low fusion protein expression.

By examining endogenous p27 expression, we concluded the endogenous p27 in

U2OS-JC17-p27-Ven cells was not responding as expected to the serum starvation, yet the endogenous

p27 in U2OS-JC13-GFP and RPE cells was responding appropriately and similarly across the

cell lines. The contrast in endogenous p27 levels across the two U2OS lines suggests that some

attribute of p27-Ven or simply its presence is influencing endogenous p27 kinetics and/or U2OS

cell cycling. Based on our findings, it is possible that the fusion protein disrupted expected

cellular events, though there are other conditions that might explain these results. One caveat to

these data is our lack of a truly successful replicate due to low lysate concentration and excessive

nonspecific antibody binding in previous replicate attempts. With this factor in mind, we

concluded that p27-Ven should not be utilized as a biosensor for endogenous p27 expression

unless further replicates prove these data incorrect.

In light of inconclusive results and time constraints, we developed novel N-terminally

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influenced by Oki et al., who created mutant N- and C-terminally tagged p27 fusion proteins,

with only the N-terminally tagged fusion functioning properly (Oki et al., 2014). We have

successfully cloned pLenti-puro-Ven-p27 and pLenti-neo-Ven-p27 constructs and are working to

generate stable hTERT-RPE1 cell lines containing these constructs. While our transduction of

hTERT-RPE1 cells with these constructs was deemed successful via immunoblot, the fusion

protein was produced at similar levels to that seen in uninduced expression of the C-terminally

tagged construct in U2OS cells. We concluded that because fluorescence was not visualized via

microscopy in the hTERT-RPE1 cells but was seen in the uninduced U2OS cells, it is possible

that a greater number of RPE cells were generating the fusion protein to a lesser extent than the

few leaky U2OS cells. Thus, we are working to generate stable cell lines that produce greater

levels of Ven-p27 and can be visualized not only via immunoblot but also via microscopy.

We hope that by changing from C- to N-terminally tagged fusions as well as from a

transformed to a non-transformed cell line, we will develop an effective biosensor system for

endogenous p27 expression. Future studies will utilize verified p27 fusion protein constructs in

mammalian cells to produce a high-resolution temporal map of protein degradation primarily via

single cell analysis using live cell imaging. With this map, the effect of protein degradation

order on cellular proliferation and genomic stability can be better understood.

Acknowledgements

I thank Dr. Jean Cook for providing helpful insight regarding modifying and approving

experimental designs and the final editing of this paper. I thank Dr. Gavin Grant not only for

designing all primers used in this study and constructing pEXPJC17-p27-Ven, but also for

serving as a mentor, training me in laboratory techniques, and assisting me in troubleshooting. I

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helpful discussion. I thank John Aster for the gift of U2OS-TRex cells, Eric Campeau for the

gift of the pLenti-neo and pLenti-puro vectors, and Jonathon Pines for the gift of the

pDESTJC13 vector. Lastly, I thank my BIOL 692H peers who reviewed this manuscript and

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References

Agrawal, D., Dong, F., Wang, Y. Z., Kayda, D., & Pledger, W. J. (1995). Regulation of cyclin E

and p27kip during mitosis in BALB/c 3T3 cells. Cell Growth & Differentiation!: The

Molecular Biology Journal of the American Association for Cancer Research, 6, 1199–

1205.

Alkarain, A., Jordan, R., & Slingerland, J. (2004). p27 deregulation in breast cancer: prognostic

significance and implications for therapy. Journal of Mammary Gland Biology and

Neoplasia, 9, 67–80. doi:10.1023/B:JOMG.0000023589.00994.5e

Bochis, O. V, Irimie, A., Pichler, M., & Neagoe, I. B.-. (2015). The Role of Skp2 and its

Substrate CDKN1B ( p27 ) in Colorectal Cancer, 24(2), 225–234.

Bonelli, P., Tuccillo, F. M., Borrelli, A., Schiattarella, A., & Buonaguro, F. M. (2014).

CDK/CCN and CDKI Alterations for Cancer Prognosis and Therapeutic Predictivity.

BioMed Research International, 2014, 361020. doi:10.1155/2014/361020

Campeau, E., Ruhl, V. E., Rodier, F., Smith, C. L., Rahmberg, B. L., Fuss, J. O., … Kaufman, P.

D. (2009). A versatile viral system for expression and depletion of proteins in mammalian

cells. PLoS ONE, 4(8). doi:10.1371/journal.pone.0006529

Chiarle, R., Budel, L. M., Skolnik, J., Frizzera, G., Chilosi, M., Corato, A., … Inghirami, G.

(2000). Increased proteasome degradation of cyclin-dependent kinase inhibitor p27 is

associated with a decreased overall survival in mantle cell lymphoma. Blood, 95, 619–626.

Chu, I. M., Hengst, L., & Slingerland, J. M. (2008). The Cdk inhibitor p27 in human cancer:

prognostic potential and relevance to anticancer therapy. Nature Reviews. Cancer, 8(4),

(28)

Coqueret, O. (2003). New roles for p21 and p27 cell-cycle inhibitors: a function for each cell

compartment? Trends in Cell Biology, 13(2), 65–70. Retrieved from

http://www.ncbi.nlm.nih.gov/pubmed/12559756

Gibson, D. G., Young, L., Chuang, R., Venter, J. C., Hutchison, C. a, & Smith, H. O. (2009).

Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods,

6(5), 343–5. doi:10.1038/nmeth.1318

Grant, G. D., Brooks, L., Zhang, X., Mahoney, J. M., Martyanov, V., Wood, T. a., … Whitfield,

M. L. (2013). Identification of cell cycle–regulated genes periodically expressed in U2OS

cells and their regulation by FOXM1 and E2F transcription factors. Molecular Biology of

the Cell, 24, 3634–3650. doi:10.1091/mbc.E13-05-0264

Hara, T., Kamura, T., Nakayama, K., Oshikawa, K., Hatakeyama, S., & Nakayama, K. I. (2001).

Degradation of p27Kip1 at the G0-G1 Transition Mediated by a Skp2-independent

Ubiquitination Pathway. Journal of Biological Chemistry, 276(52), 48937–48943.

doi:10.1074/jbc.M107274200

Hayes, O., Ramos, B., Rodríguez, L. L., Aguilar, A., Badía, T., & Castro, F. O. (2005). Cell

confluency is as efficient as serum starvation for inducing arrest in the G0/G1 phase of the

cell cycle in granulosa and fibroblast cells of cattle. Animal Reproduction Science, 87(3-4),

181–192. doi:10.1016/j.anireprosci.2004.11.011

He, W., Wang, X., Chen, L., & Guan, X. (2012). A crosstalk imbalance between p27(Kip1) and

its interacting molecules enhances breast carcinogenesis. Cancer Biotherapy &

Radiopharmaceuticals, 27(7), 399–402. doi:10.1089/cbr.2010.0802

(29)

of the interaction between p27 and Kip1 ubiquitylation-promoting complex, the ubiquitin

ligase that regulates proteolysis of p27 in G1 phase. The Journal of Biological Chemistry,

280(18), 17694–700. doi:10.1074/jbc.M500866200

Laoukili, J., Kooistra, M. R. H., Brás, A., Kauw, J., Kerkhoven, R. M., Morrison, A., …

Medema, R. H. (2005). FoxM1 is required for execution of the mitotic programme and

chromosome stability. Nature Cell Biology, 7(2), 126–136. doi:10.1038/ncb1217

Lee, J., & Kim, S. S. (2009). The function of p27 KIP1 during tumor development. Experimental

& Molecular Medicine, 41(11), 765–71. doi:10.3858/emm.2009.41.11.102

Liu, Z., Long, Y., Zhang, Y., Huang, W., Long, X., Yang, H., … Fang, W. (2013). Nuclear p27

expression confers a favorable outcome for nasopharyngeal carcinoma patients. Disease

Markers, 35(6), 925–32. Retrieved from

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3881392&tool=pmcentrez&ren

dertype=abstract

Malecki, M. J., Sanchez-Irizarry, C., Mitchell, J. L., Histen, G., Xu, M. L., Aster, J. C., &

Blacklow, S. C. (2006). Leukemia-associated mutations within the NOTCH1

heterodimerization domain fall into at least two distinct mechanistic classes. Molecular and

Cellular Biology, 26(12), 4642–4651. doi:10.1128/MCB.01655-05

Murray, A. W. (2004). Recycling the cell cycle: cyclins revisited. Cell, 116(2), 221–34.

Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/14744433

Nakayama, K., Nagahama, H., Minamishima, Y. a, Miyake, S., Ishida, N., Hatakeyama, S., …

Nakayama, K. I. (2004). Skp2-mediated degradation of p27 regulates progression into

(30)

http://www.ncbi.nlm.nih.gov/pubmed/15130491

Oki, T., Nishimura, K., Kitaura, J., Togami, K., Maehara, A., Izawa, K., … Kitamura, T. (2014).

A novel cell-cycle-indicator, mVenus-p27K−, identifies quiescent cells and visualizes G0–

G1 transition. Scientific Reports, 4. doi:10.1038/srep04012

Pellegata, N. S. (2012). MENX and MEN4. Clinics (São Paulo, Brazil), 67 Suppl 1(2), 13–8.

doi:10.6061/clinics/2012(Sup01)04

Rodier, G., Montagnoli, A., Di Marcotullio, L., Coulombe, P., Draetta, G. F., Pagano, M., &

Meloche, S. (2001). p27 cytoplasmic localization is regulated by phosphorylation on Ser10

and is not a prerequisite for its proteolysis. EMBO Journal, 20(23), 6672–6682.

doi:10.1093/emboj/20.23.6672

Shen, M., Schmitt, S., Buac, D., & Ping Dou, Q. (2013). Targeting the ubiquitin - proteasome

system for cancer therapy. Expert Opinion Therapeutic Targets, 17(9), 1091–1108.

doi:10.1517/14728222.2013.815728.Targeting

Slingerland, J. M., Hengst, L., Pan, C. H., Alexander, D., Stampfer, M. R., & Reed, S. I. (1994).

A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor

beta-arrested epithelial cells. Molecular and Cellular Biology, 14(6), 3683–94.

doi:10.1128/MCB.14.6.3683.Updated

Slingerland, J., & Pagano, M. (2000). Regulation of the Cdk Inhibitor p27 and Its, 17(October

1999), 10–17.

Vinals, F., & Pouyssegur, J. (1999). Confluence of vascular endothelial cells induces cell cycle

exit by inhibiting p42/p44 mitogen-activated protein kinase activity. Mol Cell. Biol., 19(4),

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Appendix

Resistance

Marker Forward Primer Seq 5’ to 3’ Reverse Primer Seq 5’ to 3’

pEXPJC13 -p27-Ven

DNA Sequence

hygromycin _attB-F

p27-ATGGGGGAC AAGTTTGTAC AAAAAAGCA GGCTTCATGT CAAACGTGC CGAGTGTC p27-attB-R ATGGGGGAC CACTTTGTAC AAGAAAGCT GGGTACGTT TGACGTCTTC TGAGG pEXPJC17 -p27-Ven DNA sequence

nourseothricin _attB-F

p27-ATGGGGGAC AAGTTTGTAC AAAAAAGCA GGCTTCATGT CAAACGTGC CGAGTGTC p27-attB-R ATGGGGGAC CACTTTGTAC AAGAAAGCT GGGTACGTT TGACGTCTTC TGAGG pLenti- neo-Ven-p27 DNA sequence

neomycin _Hin-FP

F-Ent-CTTTAAAGGA ACCAATTCAG TCGACAAGC TTTCGCCACC ATGGTGAGC AAGGGCGAG R-P2A-Mlu-p27 GTTAGTAGCT CCGCTTCCA CGCGTCGTT TGACGTCTTC TGAGG pLenti- puro-Ven-p27 DNA sequence

puromycin _Hin-FP

F-Ent-CTTTAAAGGA ACCAATTCAG TCGACAAGC TTTCGCCACC ATGGTGAGC AAGGGCGAG R-P2A-Mlu-p27 GTTAGTAGCT CCGCTTCCA CGCGTCGTT TGACGTCTTC TGAGG

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Figure A1. Diagram of 6-well dish layout for serum starvation experiment. “Not confluent” wells were roughly 60-80% confluent by the time of collection, whereas “confluent” wells were 100% confluent.

p27-Ven no dox 10% FBS not confluent

p27-Ven dox 10 10% FBS not confluent

p27-Ven no dox 0% FBS not confluent

p27-Ven dox 10 0% FBS not confluent

p27-Ven no dox 0% FBS confluent

p27-Ven dox 10 0% FBS confluent

p27-Ven no dox 10% FBS

confluent

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Figure A2. Immunoblot demonstrating doxycycline-inducible control of p27-Ven. By comparing U2OS-GFP and p27-Ven cell lines, we established that a nonspecific band was present on the blot after probing with p27 primary antibody. This band was

determined to be the top band because it did not increase with increasing doxycycline in either the U2OS-GFP or p27-Ven cell line and is present across both lines. Further, p27-Ven levels increase with increasing levels of doxycycline added, indicating the fusion protein is doxycycline-inducible.

Figure A3. Destination vector pcDNA5/FRT/TO-Venus-Flag-Gateway (1124)

(pDESTJC13) used for cloning pEXPJC13-p27-Ven and pEXPJC17-p27-Ven. Image generated in Vector NTI® (Thermo Scientific, DE).

pDESTJC13 7 61 7 bp

HygR(noATG) AmpR

Ve nus

ccdB CmR TATA/Te tOp2

Flag3X CM V

FRT

attR1

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Figure A4. Destination vector pLenti PGK Puro DEST (w529-2) used for cloning pLenti-puro-Ven-p27. Image generated in Vector NTI®.

Figure A5. Destination vector pLenti PGK Neo DEST (w531-1) used for cloning pLenti-neo-Ven-p27. Image generated in Vector NTI®.