Proline rich acidic protein 1 in life and death 4

Figure 3.31 PRAP1 is induced by genotoxic stressors A: Representative RT-PCR gel picture showing the expression of prap1 and gapdh in HCT 116 cells after treatment with the indicated str

induction of PRAP1 mRNA expression by serum deprivation and ethanol The common mechanism of action underlying the agents that induced PRAP1 mRNA expression was DNA damage We investigated whether PRAP1 is a genotoxic

responsive gene by using gamma irradiation, a well-characterized model to study

DNA-damage Using real-time quantitative RT-PCR, we showed that PRAP1

mRNA transcript levels were induced by gamma irradiation in a time-dependent

manner (Figure 3.31-B) The expression of PRAP1 mRNA was induced by

four-fold at four hours after exposure to gamma irradiation The response to damage peaked at six-fold, six hours after gamma irradiation These results

DNA-indicated that PRAP1 mRNA levels are regulated by DNA damage

3.5.2 Transcriptional regulation of PRAP1 by genotoxic agents

To further study the regulation of PRAP1 by the various stressors, we

selected 5-FU and CPT for our downstream studies as they induced the highest

levels of PRAP1 mRNA and are used in the treatment of colorectal cancer Firstly,

we investigated whether the regulation of PRAP1 occurs at the transcriptional level To achieve that, we first characterized the PRAP1 promoter in HCT 116 by using various PRAP1 promoter constructs as described previously under section

3.2.2.1 The majority of the promoter constructs showed an average promoter activity of 30-fold over baseline levels (without promoter) (Figure 3.32) The

longest PRAP1 promoter construct was 3900bp long and demonstrated the highest

promoter activity (50-fold) In HCT 116 cells, the core promoter was also

identified to be within 203 base pairs upstream of PRAP1 transcription start site

(Figure 3.33), similar to our previous observation in another cell line, L8 (Figure

Figure 3.31 PRAP1 is induced by genotoxic stressors

A: Representative RT-PCR gel picture showing the expression of prap1 and gapdh in HCT 116 cells after treatment with the indicated stressors (5-FU (25

µM), camptothecin (20 nM), etoposide (20 µM), serum free medium or 5% ethanol for 48 hours; hydrogen peroxide (100 µM) for 1 hour and recovered for 4 hours; and UV (20J))

B: Representative graph showing fold induction of prap1 expression in HCT 116

Figure 3.32 Luciferase assay to identify the regions required for the prap1

gene promoter activity in HCT 116 cells

Left panel: Illustration representing various portions of the prap1 5’ flanking region of the prap1 gene subcloned upstream of the firefly luciferase gene (pGL3- Basic) The number in the box indicates the size of each prap1 fragment with

respect to the transcription start site (+1)

Right panel: Representative figure showing the fold induction of prap1 promoter

activity of each fragment in HCT 116 For each transfection, the firefly luciferase activity was normalized with the Renilla reniformis luciferase activity by the cotransfected pRL-TK The relative activity of each construct is expressed as a ratio to the activity of the pGL-Basic Bar, mean of three replicates Bar, SE

Figure 3.33 Identification of core promoter of prap1 gene

Left panel: Illustration showing the deletion construct pGL (-461/-203) generated

by deleting the 203 base pairs upstream of the transcription start site from the pGL(-461/0) construct

does not seem to fully account for the marked induction of PRAP1 mRNA by the

genotoxic agents as shown in Figure 3.35, suggesting the possible involvement of other transcriptional regulatory mechanisms such as cis- or trans-regulatory

elements and/or stability of mRNA

3.5.3 Regulation of PRAP1 protein by genotoxic agents

In order to study the intracellular level of PRAP1, which is a highly secreted protein (Zhang, Wong et al 2003), we used Brefeldin A (BFA), a lactone antibiotic that inhibits the protein transport from the endoplasmic reticulum (ER)

to the Golgi apparatus, to trap PRAP1 in the ER (Pelham 1991; Klausner, Donaldson et al 1992) After exposing HCT 116 cells to 5-FU and CPT for 48 hours, PRAP1 was trapped in the ER with BFA for 6 hours before harvesting As shown in Figure 3.36, PRAP1 protein was highly induced by both 5-FU and CPT

at various dosages which corresponded to the increase observed at the mRNA level 5-FU induced a higher level of PRAP1 protein expression as compared to that of the CPT at all doses The induction of PRAP1 by CPT was dose-dependent whereas the induction of PRAP1 by 5-FU was saturated at the lowest dosage used

Figure 3.34 Induction of prap1 promoter activity by 5-FU and CPT

The longest promoter construct 3900) and core promoter construct 203) were cotransfected with pRL-TK into HCT 116 cells for 24 hours 5-fluorouracil, 25µM of 5-FU (A) and 20nM of camptothecin, CPT (B) was added

(pGL-to the cells for another 24 hours The firefly luciferase activity was normalized with the Renilla reniformis luciferase activity Promoter activity of each construct was expressed as fold induction relative to that of the pGL3-Basic Column, mean

of three replicates; Bar, SE

Figure 3.35 Induction of PRAP1 by 5-FU and CPT at mRNA level

Representative RT-PCR gel picture showing the mRNA expression of prap1 and gapdh in the cells from Figure 3.34

Figure 3.36 PRAP1 was induced at protein level by 5-FU and CPT

Representative western blot of PRAP1 and GAPDH in HCT116 cells after treatment with the indicated doses of either 5-fluorouracil (5-FU) or camptothecin, (CPT) or untreated (control) for 48 hours Brefeldin A was added to trap the PRAP1 protein in endoplasmic reticulum for 6 hours before harvesting Cytoplasmic protein (100 µg) was subjected to western blot analysis with a specific anti-PRAP1 antibody The membrane was reprobed with anti-GAPDH antibody to confirm equal loading

3.5.4 Dose- and time-dependent regulation of PRAP1

To study the dose and time response of PRAP1 mRNA level by 5-FU and

CPT, HCT 116 cells were treated with different doses of 5-FU (2.5 and 25 µM) and CPT (20 and 250nM) for 3 days Cells were harvested for RT-PCR and

western blot analysis at 24, 48 and 72 hours The expression of PRAP1 mRNA

was induced as early as 24 hours and sustained at 72 hours when treated with both low and high doses of 5-FU (Figure 3.37) In the presence of low dose of CPT,

PRAP1 mRNA was induced at 24 hours and peaked at 48 hours, whereas with high dose of CPT, the expression of PRAP1 mRNA peaked at 24 hours

The time-course regulation of PRAP1 expression at protein level was studied over 72 hours using high doses of 5-FU or CPT The induction of PRAP1 protein by 5-FU occurred as early as 24 hours and peaked at 48 hours (Figure 3.38), whereas the induction of PRAP1 by CPT peaked at 24 hours This temporal regulation of PRAP1 by genotoxic stress suggests that it has a specific temporal function in the cellular response to genotoxic stress

3.6 Wild-type-p53-dependent induction of PRAP1

3.6.1 Genotoxic agents failed to induce PRAP1 in p53-/- cells

As p53 plays an important role in the response to DNA damage (Sionov

and Haupt 1999), we investigated whether p53 is a regulator of PRAP1 expression

upon exposure to DNA damage agents We used two cell lines derived from the parental HCT 116 cells, one with p53 knockout (p53-/-) and one with p21

knockout (p21-/-) PRAP1 mRNA was induced in both HCT 116 wild-type and

Figure 3.37 Early upregulation of PRAP1 in a dose- and time-dependent

manner

Representative RT-PCR gel picture of prap1 and gapdh in HCT 116 cells after

treatment with 5-FU and CPT at the indicated doses for 24, 48 and 72 hours

Figure 3.38 Early upregulation of PRAP1 protein by 5-FU and CPT

Representative western blot of PRAP1 and GAPDH in HCT116 cells after

treatment with 5-FU (25µM) and CPT (20nM) for 24, 48 and 72 hours Brefeldin

A was added to trap the PRAP1 protein in endoplasmic reticulum for 6 hours

before each harvesting Cytoplasmic protein (100 µg) was subjected to immunoblot analysis with a specific anti-PRAP1 antibody The membrane was

reprobed with anti-GAPDH antibody to confirm equal loading

cells at both low and high doses of 5-FU and CPT (Figure 3.39) These results

suggested that induction of PRAP1 by DNA damaging agents was dependent on

p53

3.6.2 Restoration of PRAP1 induction by reintroduction of wild-type p53 in

p53-/- cells

To confirm that the induction of PRAP1 was dependent on p53, we

reintroduced the wild type p53, pCMV-p53 (WT) or the DNA-binding deficient mutant p53, pCMV-p53mt153 (Mut) into p53-/- cells Our results showed that, the

PRAP1 mRNA was detectable in WT-transfected p53-/- cells, but not in Mut or empty vector-transfected p53-/- cells, suggesting that PRAP1 may be a direct target gene of p53 In the presence of 5-FU or CPT, the expression of PRAP1

mRNA was further induced in the WT-transfected p53-/- cells, but not in the Mut

or vector-transfected cells (Figure 3.40) This indicated that the induction of

PRAP1 requires the expression of wild-type p53 While p53 is best characterized

as a transcription factor, the data presented here does not distinguish between the

following two possible roles of p53 in the induction of PRAP1 expression following exposure to DNA damaging agents Firstly, PRAP1 may be a direct or

indirect transcriptional target of p53 Secondly, p53 which is a DNA binding

protein, may displace an unknown repressor from the PRAP1 promoter

3.6.3 Genotoxic agents failed to induce PRAP1 in Hep 3B and HT 29 cells

To further validate that wild-type p53 is required for the induction of

PRAP1 expression by genotoxic agents, we used two cell lines with inherent p53 defects: HT 29, a cell line with transcriptional mutant p53 (HT 29) and Hep 3B, a

Figure 3.39 Induction of prap1 by 5-FU and CPT is dependent on p53

Representative RT-PCR gel picture of prap1 and gapdh in HCT116 cells and its

two derivatives, p53-/- and p21-/- after treatment with the indicated doses of 5-FU and CPT for 24 hours

Figure 3.40 Reintroduction of wild-type p53 rescues the induction of prap1 by

5-FU and CPT in p53-/- cells

Representative RT-PCR gel picture of prap1 and gapdh in p53-/- cells after

transfection with either empty vector control (V) or wild-type p53 (WT) or mutant p53 (Mut), followed by treatment with either 5-FU (25µM) or CPT (20nM) for 24 hours

cell line with p53 deficiency Consistent with our observation, both 5-FU and CPT

failed to induce PRAP1 mRNA expression in HT 29 and Hep 3B cells as

compared to their respective p53 wild-type cell lines, HCT 116 and Hep G2

(Figure 3.41) These results support the conclusion that the induction of PRAP1

by DNA damage agents is dependent on wild-type p53

3.7 PRAP1 is a novel p53-responsive gene

3.7.1 Identification of p53-response elements in PRAP1 gene

Following confirmation that induction of PRAP1 is dependent on type p53, we investigated whether p53 was able to regulate PRAP1 directly at the

wild-transcriptional level Using an algorithm designed to search for potential p53 binding sites (Hoh, Jin et al 2002), we identified two potential p53-responsive

elements within intron 1 of PRAP1 The sequences of these two p53 responsive elements in PRAP1 matched the consensus p53 binding site used by the algorithm

by 84% and 70% (Table 3.11) In comparison, the known p53-response element in p21 promoter showed a match of 90% The positions of the two p53-responsive

elements (+1316 and +1460) in PRAP1 are illustrated in Figure 3.42

In order to determine whether the two identified p53-response elements in

PRAP1 (PRAP1-p53BS) are functional, we amplified the intronic fragment from positions +1197 to +1534 which contains both p53-response elements of PRAP1

This fragment was cloned into a pGL3-promoter luciferase reporter vector upstream of a minimal SV-40 promoter as illustrated in Figure 3.43 The constructed plasmid (pGL3-PRAP) was checked by restriction enzyme digestion (insert size, 337bp; Figure 3.44) and verified by sequencing

Figure 3.41 Hep 3B and HT 29 cells failed to induce prap1 gene expression

Representative RT-PCR gel picture of prap1 and gapdh in HCT 116 (p53 WT)

versus HT 29 (p53 mutant) and Hep G2 (p53 WT) versus Hep 3B (p53 deficient) cells after treatment with 5-FU (25µM) and CPT (20nM) for 48 hours

Figure 3.42 Schematic diagram of PRAP1 gene

Two p53 binding sites were identified in intron 1 of PRAP1 Their distribution and position was showed in the diagram with transcription start site of PRAP1 gene denoted as +1

Table 3.11 Sequences of the two p53 binding sites located in PRAP1 gene

The consensus sequence of p53 was showed in the second row of the table The p53 binding site located in p21 was included as positive control for comparison Row three and four indicate the sequences of p53 binding site in PRAP1 gene as predicted by the algorithm

were cloned into the luciferase reporter plasmid (pGL3-Promoter) before the SV

40 promoter as showed in the diagram

Figure 3.44 Verification of the p53 binding sites constructed plasmid

Representative DNA agarose gel picture of the amplified insert in the constructed plasmid (PRAP1) and the empty plasmid (Vector) A size difference of about 300bp was observed that indicated the presence of p53 binding sites

3.7.2 p53-response elements in PRAP1 gene are responsive to wild-type p53

To examine the responsiveness of these two potential p53-response

elements in intron 1 of PRAP1, the pGL3-PRAP construct was transiently

cotransfected with empty pCMV (Vector), p53 (p53 WT) or p53mt153 (p53 Mut) into p53-/- cells In this study, the p53-response element in p21 (pGL3-p21) was used as a positive control for comparison The empty pGL3-Promoter vector was used as negative control We found that transfection of p53

pCMV-WT, but not Vector or p53 Mut, increased the SV-40 promoter activities by about three-fold in cells transfected with pGL3-PRAP (Figure 3.45) The extent of the

enhanced SV-40 promoter activity by the two p53-response elements in PRAP1

gene was comparable to that of the p21 These results demonstrated that the

p53-response elements in PRAP1 were functionally as effective as that in p21 in

mediating p53-induced gene transcriptional activation

3.8 PRAP1 modulates cell fate after genotoxic stress

3.8.1 Repression of PRAP1 induction by siRNAs

After p53 activation upon DNA damage, mammalian cells either execute cell cycle arrest, senescence or apoptosis To investigate the biological functions

of p53 mediated PRAP1 induction, we determined the effects of PRAP1 inhibition

by siRNA on p53-induced cellular response such as apoptosis To achieve this, we

used two custom-made synthetic siRNAs that target PRAP1 (PRAP1 siRNA1 and PRAP1 siRNA2) These sequences are verified to be PRAP1-specific using

BLAST from NCBI Web Page To account for the non-specific effect of siRNA,

Figure 3.45 Predicted p53 binding elements of PRAP1 response to wild-type p53

Representative figure showing the fold induction of SV40 promoter activity of pGL3-P (basic vector), pGL3-PRAP and pGL3-p21 cotransfected with pcDNA vector, pcDNA-p53 and pcDNA-p53Mut in p53-/- cells for 48 hours For each transfection, the firefly luciferase activity was normalized with the Renilla reniformis luciferase activity by the cotransfected pRL-TK The relative activity

of each construct is expressed as a ratio to the activity of the pGL3-P pGL3-P, Basic luciferase promoter; pGL3-PRAP, Basic luciferase promoter plasmid constructed with the two p53 binding elements of PRAP1 gene; pGL3-p21, Basic luciferase promoter plasmid constructed with the p53 binding elements of p21 gene (serving as a positive control for comparison); Vector: pcDNA vector; p53 WT: pcDNA with wild type p53 construct; p53 Mut: pcDNA with mutant p53

siRNA2) These sequence-specific control siRNAs do not target any mammalian

genes or PRAP1 gene

HCT 116 cells were plated for 24 hours and cells were transfected with PRAP1 siRNAs or control siRNAs 48 hours after siRNA transfection, these cells were treated with 5-FU for another 24 hours prior to harvesting for mRNA and protein analysis The PRAP1 induced by 5-FU was effectively repressed by PRAP1 siRNAs at both mRNA (Figure 3.46) and protein level (Figure 3.47) as compared to their respective control siRNAs PRAP1 siRNA1 has a greater silencing effect than that of PRAP1 siRNA2 The sequence-specific control

siRNAs did not target the PRAP1 gene These results indicated that we have

generated an effective and specific siRNA system to repress e 5-FU induced PRAP1 expression

3.8.2 Effect of PRAP1 knockdown on colony formation

The effect of PRAP1 repression on the efficacy of 5-FU in inhibiting the establishment and growth of colonies in HCT 116 cells was studied over a period

of 7 days Our results showed that PRAP1 knockdown cells exhibited a significant reduction in their size and the number of colonies formed as compared to their respective control siRNAs (Figure 3.48) There was about 50% reduction in the number of colonies formed in cells with PRAP1 knockdown treated with 3 µM 5-

FU as compared to that of control siRNAs At a higher dose of 5-FU (6 µM), the number of colonies was reduced by 80% (Figure 3.49) These results indicated that the inhibition of the induced PRAP1 expression by 5-FU enhanced the anti-tumor effect of 5-FU, suggesting that expression of PRAP1 is able to modulate the

Representative RT-PCR agarose gel picture of prap1 and gapdh in HCT 116 after

transfection with PRAP1 specific siRNA (PRAP1 siRNA1 and 2) or PRAP1 siRNA specific control siRNA (control siRNA1 and 2) for 48 hours, and followed

by the addition of 5-FU for another 24 hours

Figure 3.47 Suppression of PRAP1 induction by 5-FU at protein level

Representative western blot of PRAP1 and GAPDH in HCT 116 after transfection with PRAP1 specific siRNA (PRAP1 siRNA1 and 2) or PRAP1 siRNA specific control siRNA (control siRNA1 and 2) for 48 hours, and followed by the addition

of 5-FU for another 24 hours Brefeldin A was added 6 hours before harvesting to prevent PRAP1 from being secreted by trapping it in endoplasmic reticulum A total of 100μg of cytoplasmic protein was loaded for the analysis

Figure 3.48 Repression of PRAP1 expression reduces colony numbers

Representative result of colony formation assay performed on HCT116 cells The cells were transfected with either control siRNAs or PRAP1 siRNAs After 48 hours, cells were treated with 5-FU for 24 hours Cells were then replated and maintained for 7 days in culture for colony formation Colonies were stained with crystal violet for visualization Representative images were taken

Figure 3.49 Summary of the number of colonies formed

Total number of colonies for each assay were counted using ImageJ A statistical analysis of the colony formation assay was performed Column, mean of 3

replicates; Bars, SEM * p<0.05