Role of the survival proteins hsp27 and survivin in a small molecule sensitization to TRAIL mediated apoptosis 2

Dose response curves for TRAIL and LY30 were established in HeLa cells by evaluating the effect of increasing doses of TRAIL 0 to 250 ng/mL and LY30 0 to 25 µM, alone or in combination,

RESULTS

LY30 restores HeLa cells sensitivity to TRAIL-induced cell death

Dose response curves for TRAIL and LY30 were established in HeLa cells by evaluating the effect of increasing doses of TRAIL (0 to 250 ng/mL) and LY30 (0 to

25 µM), alone or in combination, on cell viability after 16h by using the crystal violet assay (Figure 13A) HeLa cells did not exhibit a decrease in cell viability upon TRAIL treatment even at the highest dose used (250 ng/mL) However, pre-incubation with different doses of LY30 for 1h significantly reduced cell viability (50% viability with 25 !M LY30 and 20 ng/mL TRAIL in combination) as compared

to both treatment alone (85% viability for 25 µM LY30 and 100% viability for 20 ng/mL), hence corroborating our earlier finding that LY30 is able to sensitize HeLa cells to TRAIL-induced cell death [156] Interestingly, the observed sensitization was enhanced in a dose dependent manner as a function of TRAIL dose but not of LY30 dose Further cell viability assays for TRAIL treatment alone were carried out over a longer time frame (up to 72h) (Figure 13B) HeLa cells viability was not decreased upon treatment with TRAIL for a longer period Only HeLa cells treated with 250 ng/mL of TRAIL for 72h showed a modest effect of TRAIL on cell viability

For subsequent experiments, the chosen doses of LY30 and TRAIL were 25

LY30 and TRAIL combined treatment decreases HeLa cells ability to form colonies

Given the ability of LY30 and TRAIL combination to induce cell death upon short-term treatment, we were interested in understanding the effect of LY30+TRAIL

on the long-term survival/proliferative capacity of tumor cells This was done by evaluating the effect of the combined drug treatment on HeLa cells colony forming ability

HeLa cells were exposed to LY30 for 1hr before incubation with TRAIL for 6h An equal number of cells were then seeded onto 100mm Petri dishes and allowed

to form colonies over a period of 10 to 14 days

After staining the cells with crystal violet, we observed a marked reduction in the number of colonies formed for cells treated with both compounds (Figure 14A) A significant decrease in clonogenic ability (35% of untreated cells) was observed for cells under combinatorial treatment as compared to cells treated singly with either TRAIL or LY30 (Figure 14B) Interestingly, TRAIL alone, and to a lesser extent LY30 alone, induced a significant decrease of the size of the colonies (40% when compared to control) (Figure 14C) The combined treatment further reduced the size

of the colonies This observation explains the discrepancy between the actual number

of colonies counted after TRAIL treatment and the apparent low number of colonies

in the picture itself

Figure 13: Effect of LY30 treatment on TRAIL-mediated apoptosis

Hela cells exposed to TRAIL (0-250 ng/mL) for 16h with or without 1h

pre-Figure 14: Effect of LY30 and TRAIL treatment on colony forming ability of HeLa cells

HeLa cells were exposed to 20 ng/ml TRAIL for 6h with or without pre-incubation with 25 !M LY30 The cells were then re-seeded onto 100 mm Petri dishes and allowed to form colonies over 10 to 14 days, followed by staining with crystal violet

(A) for colony counts (B) and colony size (C) determination Data shown are the mean±S.D of three independent experiments *p<0.01

LY30, alone or in combination with TRAIL, induces an early mitochondrial membrane potential depolarization (!" m) and mitochondrial aggregation

It has been shown that mitochondrial outer membrane permeability (MOMP) involves a drop in the mitochondrial transmembrane potential (!"m) We set out to investigate the effect of the combined treatment of TRAIL and LY30 on !"m

HeLa cells were treated with TRAIL for 1h to 4h with or without incubation with LY30 for 1h followed by !"m analysis with TMRE by laser scanning cytometry The uncoupler CiCCP was used as a positive control for depolarization (Figure 15A)

pre-Results show that LY30, alone or in combination with TRAIL, was able to induce a drop of the mitochondrial transmembrane potential in a time-dependent manner (Figure 15B) In addition, an alternate analysis of the data for the 4h time point indicated that LY30+TRAIL treatment, and to a lesser extent LY30 alone, was inducing an early mitochondrial aggregation (Figure 16A and B) – a phenomenon shown to occur prior MOMP [418]

Figure 15: Effect of LY30 and TRAIL treatment on mitochondrial membrane polarization

HeLa cells were treated for 1h to 4h with 20 ng/mL of TRAIL with or without incubation with 25 µM LY30 Live cells were then incubated with TMRE and Hoescht before analysis by Laser Scanning Cytometry The uncoupler CiCCP (5 !M)

pre-was used as a positive control for depolarization of the mitochondrial membrane (A) Histogram representing mitochondrial membrane polarization (B) n=2

Figure 16: Effect of upon LY30 and TRAIL treatment on mitochondrial aggregation

(HeLa cells were treated for 4h with 20 ng/mL of TRAIL with or without

pre-incubation with 25 !M LY30 Live cells were then incubated with TMRE and

LY30 and TRAIL treatment engages the mitochondrial apoptotic pathway

The release of apoptogenic factors like cytochrome c or Smac/DIABLO (cyt c and Smac) from the mitochondria intermembrane spaces marks an important step in the intrinsic apoptotic pathway as it engages the apoptotic cascade Earlier results showed that LY30 combined to TRAIL is able to induce MOMP, as evidenced by a drop in !"m as well as mitochondrial aggregation, hence we set out to investigated subsequent mitochondrial events such as the release of apoptogenic factors

HeLa cells were treated with TRAIL for 6h, 12h or 18h with or without incubation with LY30 for 1h followed by subcellular fractionation and analysis of cyt

pre-c and Smapre-c release into the pre-cytosol by Western blotting The purity of the pre-cytosolipre-c and mitochondrial fraction was confirmed by monitoring the absence of VDAC and CuZnSOD in the cytosolic or mitochondrial fraction, respectively

As seen in Figure 17, both cytochrome c and Smac were detected in the cytosolic fraction after 12h upon LY30 and TRAIL treatment At 18h, both proteins were found in greater amount in the cytosol, a phenomenon coupled with a decrease

in the level of both proteins in the mitochondrial fraction

These data indicate that LY30-mediated sensitization to TRAIL-induced cell death engages the mitochondrial apoptotic pathway

Figure 17: Effect of LY30 and TRAIL treatment on cytochrome c and Smac release from the mitochondria

Hela cells were exposed to 20 ng/ml TRAIL for 6h, 12h or 18h with or without incubation with 25 µM LY30 for 1h Levels of cytochrome c and Smac in the mitochondrial and cytosolic fractions were then assayed by western blotting

pre-LY30 and TRAIL combined treatment induces, and is dependent on, caspase activation

Death receptor mediated apoptosis requires the activation of the caspase cascade, which is responsible for the cleavage of cellular substrates to bring about the controlled demise of cells In order to find out whether caspase were involved in LY30 sensitization to TRAIL-mediated cell death in our cell system, we set out to investigate the activation of caspases in HeLa cells

Cells were treated with TRAIL for different time (6h, 12h and 18h) The activity of caspase-3, caspase-8 and caspase-9 was then assessed by caspase activity assay with the respective fluorescence-conjugated caspase substrates (DEVD-AFC, IETD-AFC and LEHD-AFC) Pre-treatment of HeLa cells with LY30 followed by TRAIL treatment resulted in a strong amplification of caspase-3, -8 and -9 activities

as compared to untreated cells, with a peak of activity observed at 12h for all three caspases (Figure 18A, B and C) TRAIL treatment alone was unable to induce the activation of any of the caspases investigated and exposure of HeLa cells to LY30 resulted in minimal caspase activation (less than 2 fold increase) after 6h and 12h of treatment However, LY30 treatment resulted in an increased caspase-8 and -9 activity at 18h In addition, we observed the appearance of a 17 kDa cleaved fragment

of caspase-3 at 8h following treatment with LY30 and TRAIL (Figure 18D), indicating an activation of pro-caspase-3 via proteolytic cleavage The caspase-3-mediated cleavage product of one of the caspase-3 substrate, the DNA repair enzyme PARP, was also detectable at 8h At 20h, the 17 kDa cleaved fragment of caspase-3 can observed following LY30 treatment, alone or in combination with TRAIL However, unlike LY30 treatment alone, the combined treatment resulted in a marked

and up to 18h This observation was confirmed by the complete cleavage of PARP after 18h of the combined treatment, as opposed to the incomplete cleavage observed for the treatment with LY30 alone

In order to validate the involvement of caspase-mediated cell death in our system, we investigated the effect of the pan-caspase inhibitor zVAD-fmk, as well as the effect of specific inhibitors, on the observed caspase activation The activation of all three caspases resulting from the combined treatment of TRAIL and LY30 was blocked by zVAD-fmk at 12h (Figure 18A, B and C) Moreover, specific inhibitors for caspase-3 and -8 activity (z-DEVD-fmk and z-IETD-fmk, respectively) showed similar results However, the caspase-9 inhibitor (z-LEHD-fmk) was unable to block caspase-9 acitvity as efficiently as the other inhibitors, and therefore was not used in subsequent experiments In addition, z-VAD-fmk, z-DEVD-fmk and z-IETD-fmk were able to rescue HeLa cells exposed to LY30+TRAIL treatment (Figure 19)

Taken together, these results show that the combination of LY30 and TRAIL induces a caspase-dependent apoptotic cell death in HeLa cells

Figure 18: Effect of LY30 and TRAIL treatment on caspase activation

HeLa cells were exposed to 20 ng/ml TRAIL for 6, 12 or 18h with or without treatment with 25 !M LY30 for 1h, in the presence or absence of z-VAD-fmk, z-DEVD-fmk, z-IETD-fmk or z-LEHD-fmk (50 !M each) Whole cell lysates were used to determine the activities of caspase-3, -8 and-9 using fluorescent-conjugated substrates Data shown are the mean±S.D of three independent experiments

pre-(A)(B)(C) HeLa cells were exposed to 20 ng/ml TRAIL for 4h, 8h or 20h with or

without pre-treatment with 25 !M LY30 for 1h Whole cell lysates were then used to

assay for caspase-3 processing by Western blotting (D) *p<0.01

Figure 19: Effect of caspases inhibitors on cell viability upon LY30 and TRAIL treatment

HeLa cells were exposed to 20 ng/ml TRAIL for 16h with or without pre-incubation with 25 !M LY30 for 1h, in the presence or absence of z-VAD-fmk, z-DEVD-fmk, z-IETD-fmk or z-LEHD-fmk (50 !M each) The percentage of cell survival was determined by crystal violet assay Data shown are the mean±S.D of three independent experiments *p<0.01

LY30 and LY30+TRAIL treatment decrease Hsp27 protein level in cleared RIPA cell lysates

Several studies have previously shown that both quercetin and LY29 (which is derived from quercetin) were be able to regulate Hsp27 expression at the transcription level by interfering with Hsp27 primary transcription factor Hsf-1 [294, 413-415] Since LY30 is derived from LY29, we decided to investigate whether LY30, alone or

in combination with TRAIL, also had an effect on Hsp27 expression

HeLa cells were treated with TRAIL for 20h with or without pre-incubation with LY30 for 1h followed by cell lysis in RIPA buffer Cell lysates were then cleared (i.e centrifuged) and the supernatants were analyzed by western blotting

Results show that LY30 treatment, alone or in combination with TRAIL, induced a decrease in Hsp27 protein levels in cleared RIPA lysates after 20h as evidenced by Western blot analysis (Figure 20) However, no change could be observed in Hsp70 or Hsp90 protein levels, suggesting that LY30 had an effect specifically on Hsp27

Figure 20: Effect of LY30 and TRAIL on Hsp27 and other Hsps expression

HeLa cells were exposed to 20 ng/ml TRAIL for 20h with or without pre-incubation with 25 !M LY30 for 1h Levels of Hsp27, Hsp70 and Hsp90 in the cell lysates were then assayed by Western blotting Actin was used as a control for equal loading between the wells

LY30-mediated decrease in Hsp27 protein level is not due to transcriptional regulation nor proteasomal degradation

Intrigued by this reduction in Hsp27 protein level upon LY30 treatment, we decided to examine Hsp27 mRNA expression as well the effect of the inhibition of proteasomal degradation

HeLa cells were treated for 4h to 24h with TRAIL with or without incubation with LY30 followed by total RNA extraction, reverse-transcription and PCR using primers specific for hsp27

pre-Interestingly, no decrease in Hsp27 total RNA could be detected at any of the time points tested (Figure 21A), indicating that LY30-mediated decrease in Hsp27 expression is not occurring through transcriptional regulation

Surprisingly, HeLa cells pre-treated with the proteasomal inhibitor lactacystin prior exposure to TRAIL, alone or in combination with LY30, showed a similar decrease in Hsp27 protein level as the one observed in cell untreated with lactacystin (Figure 21B)

Taken together, these results indicate that the decrease in Hsp27 protein level observed upon LY30 treatment is not due to transcriptional regulation of Hsp27 nor is

it due to an increase in Hsp27 protein degradation

Figure 21: Effect of LY30 treatment on Hsp27 transcriptional regulation and proteasomal degradation

HeLa cells were exposed to 20 ng/ml TRAIL for 4h, 8h, 12h, 18h or 24h with or without pre-incubation with 25 !M LY30 for 1h Total RNA levels of Hsp27 was

then determined by PCR Actin was used as a control for equal loading (A) HeLa

cells were exposed to 20 ng/ml TRAIL for 16h with or without pre-incubation with 25

!M LY30 for 1h in the presence or absence of 10 !M lactacystin Hsp27 protein level

in cell lysates was then assayed by Western blotting (B)

LY30, alone or in combination with TRAIL, does not affect Hsp27 expression but instead induces a long lasting translocation of Hsp27 to a nuclei-enriched fraction

As the previous results showed, the decrease in Hsp27 protein level upon LY30 treatment is not due to transcriptional regulation or increased protein degradation Interestingly, Hsp27 is known to translocate to the nucleus and/or to form high molecular size aggregates upon certain cellular stresses Hence, if the cell lysis was incomplete with the RIPA buffer used, a significant fraction of total Hsp27 protein could be lost during the clearing of the samples Similarly, high molecular size aggregates, with little solubility, could remain in the pellet In order to test this hypothesis, we decided to analyze the two fractions (supernatant and pellet) obtained after cell lysis in RIPA and clearing by centrifugation

HeLa cells were treated with TRAIL for 6h or 16h, with or without incubation with LY30 for 1h The cells were lysed using RIPA buffer and the lysates were centrifuged Both the supernatant and the pellet (resuspended in RIPA buffer) were analyzed by Western blotting

pre-As previously shown, there was a marked reduction of Hsp27 protein level in the supernatant fraction after 16h of treatment with LY30 and, to a greater extent, in the cells treated with both drugs This decrease in the supernatant fraction was mirrored by an increase in Hsp27 protein level in the nuclei-enriched pellet Such an increase was also observed in the supernatant fraction at 6h in the LY30+TRAIL treated cells although to a lesser extent (Figure 22A)

After showing that Hsp27 was able to translocate to the pellet fraction after

Hsp27 expression To that end, HeLa cells were treated with TRAIL for 4h, 8h or 20h with or without pre-incubation with LY30 for 1h followed by cell lysis in RIPA buffer Whole cell lysates (i.e no clearing by centrifugation) were then analyzed by western blotting As seen in Figure 22B, no change in Hsp27 expression at the protein level could be observed for any of the treatments or any of the time points when whole cell lysates were used instead of cleared lysates This result indicates that LY30 does not have an effect on Hsp27 expression but is able to influence Hsp27 localization in the cell

To clear out any doubt about an incomplete cell lysis being responsible for the apparent decrease in Hsp27 protein level upon treatment with LY30, we analyzed samples that were sonicated prior to centrifugation in order to insure a complete lysis

As seen in Figure 22C, no more Hsp27 could be detected in the pellet fraction coming from sonicated cells, confirming that the cell lysis was only partial

As stated above, Hsp27 translocation could be observed after 6h of treatment and was even more pronounced after 16h Interestingly, HeLa cells exposed to a 1h sub-lethal heat-shock showed a much faster nuclear translocation of Hsp27 (Figure 23) as most Hsp27 protein was detected in the nucleus after 1h However, as soon as the cells were allowed to recover at 37°C, Hsp27 rapidly translocated back into the cytoplasmic compartment and could no longer be detected in the nucleus after 6h of recovery This indicates that LY30-induced nuclear translocation of Hsp27 is a slow

protein level In addition, it indicates that Hsp27, upon LY30 treatment, translocates

to a nuclei-enriched pellet fraction

Figure 22: Effect of LY30 and TRAIL treatment on Hsp27 cellular localization

HeLa cells were treated for 6 or 16h as described earlier Hsp27 expression in the

Figure 23: Effect of heat-shock on Hsp27 cellular localization

HeLa cells were heat-shocked at 44°C for 1h and allowed to recover at 37°C for up to 6h Hsp27 levels in the nuclear and cytosolic fraction at different times before and after the heat-shock were then assayed by Western blotting

Hsp27 specifically translocates to the nucleus upon exposure to LY30

In order to ascertain that Hsp27 was translocated to the nucleus, we set out to monitor the presence of Hsp27 in different cellular fractions obtained by using classical cellular fractionation methods

HeLa cells were treated with TRAIL for 16h with or without pre-incubation with LY30 for 1h followed by either mitochondrial or nuclear subcellular fractionation and analysis by Western blotting

As seen in Figure 24A, Hsp27 protein level decreases in the cytosolic fraction after treatment with LY30 but concomitantly increases in the nuclei-enriched fraction, hence confirming previous results Moreover, no change in Hsp27 protein level can

be observed in the mitochondrial fraction In addition, a classical nuclear fractionation led to similar results (Figure 24B), confirming that Hsp27 does indeed specifically translocate to the nucleus after treatment with LY30

Figure 24: Nuclear translocation of Hsp27 upon LY30 and TRAIL treatment

Hela cells were treated for 16h with 20 ng/mL TRAIL with or without pre-incubation with 25 !M LY30 for 1h Hsp27 levels in the mitochondrial, cytosolic and nuclear

fraction obtained after cell fractionation (A) or specific nuclear fractionation (B) were

then assayed by Western blotting

Hsp27 phosphorylation increases upon exposure to LY30

After confirming that LY30 could induce Hsp27 translocation to the nucleus,

we were interested to find out the mechanisms involved in the observed translocation

As stated earlier, Hsp27 is known to be able to translocate to the nucleus when cells are exposed to certain stresses Even though the precise mechanism by which Hsp27 enters or leaves the nucleus is still unknown, it is commonly accepted that it occurs via passive transport through nuclear pores Therefore, the size of Hsp27, or rather Hsp27 oligomers, has to be small enough in order to permit entry into the nucleus Since Hsp27 oligomerization is dependent on its phosphorylation, we first set out to analyze the phosphorylation status of Hsp27 upon treatment with LY30 and TRAIL

HeLa cells were treated with TRAIL, with or without pre-incubation with LY30 for 1h, for different times followed by analysis by Western blot

As seen in Figure 25A, a marked increase in phosphorylation of Hsp27 on Ser-82 residue can be observed as early as 30 minutes after addition of LY30 in the medium Hsp27 phosphorylation reached a peak between 1h and 3h of treatment before decreasing slightly Interestingly, Hsp27 phosphorylation remained high up to 16h In addition, it can be noted that TRAIL alone does not induce Hsp27 phosphorylation but is able to enhance the phosphorylation induced by LY30 to some extent (Figure 25B)

Interestingly, in the supernatant and pellet fractions of cells treated for 16h

(Figure 25C) These results also indicate that most of the fraction of Hsp27 that translocates to the nucleus is phosphorylated

Figure 25: Effect of LY30 treatment on Hsp27 phosphorylation status

HeLa cells were treated with 25 !M LY30 for the indicated times Total Hsp27 and phospho-Hsp27 (ser82) levels in the whole cell lysates were then assayed by Western

blotting (A) Hela cells were treated with 20 ng/mL TRAIL with or without

pre-incubation with 25 !M LY30 for 1h Total Hsp27 and phospho-Hsp27 (Ser-82) levels

in the whole cell lysates were then assayed by Western blotting (B) Hela cells were

treated with 20 ng/mL TRAIL with or without pre-incubation with 25 !M LY30 for 1h Total Hsp27 and phospho-Hsp27 (ser82) levels in the pellet and supernatant

fractions were then assayed by Western blotting (C)

Hsp27 increased phosphorylation is dependent on LY30-mediated p38 protein kinase activation

Previous results showed that LY30 induces a rapid and long lasting phosphorylation of Hsp27 It has been shown that Hsp27 phosphorylation on Ser-82 residue is preferentially regulated by MAPKAP kinase 2 through p38 MAPK Hence,

we set out to investigate the role of p38 MAPK in LY30-mediated phosphorylation of Hsp27

HeLa cells were treated with TRAIL, with or without LY30 pre-incubation, for 6h followed by analysis of Hsp27 phosphorylation on Ser82 residue by western blotting As a control, we used a specific inhibitor of p38 MAPK, SB203580

As expected from previous results, LY30, alone or in combination with TRAIL, induced a strong phosphorylation of Hsp27 on Ser-82 residue after 6h of treatment However, pre-treatment with SB203580 drastically inhibited LY30-mediated phosphorylation, suggesting that p38 MAPK is involved in this process (Figure 26)

Figure 26: Effect of LY30 treatment on p38 MAPK activity

Hela cells were treated for 16h with 20ng/mL TRAIL with or without pre-incubation with 25 !M LY30 for 1h, in the presence or absence of 10 !M SB205380 Total Hsp27 and phospho-Hsp27 (Ser-82) levels in the whole cell lysates were then assayed

by Western blotting (A) HeLa cells were treated as indicated p38 MAPK activity

was then assayed as described in the Material and Methods section Data shown are

the mean±S.D of three independent experiments (B)

Hsp27 increased phosphorylation is dependent on LY30-mediated Protein Phosphatase 2A (PP2A) inhibition

In addition to the MAPKAP kinase 2/p38 MAPK, Hsp27 phosphorylation can

be modulated by the Protein Phosphatase 2A Consequently, we set out to investigate the role of protein phosphatase, and more particularly PP2A, in LY30-mediated phosphorylation of Hsp27

Again, HeLa cells were treated with TRAIL, with or without LY30 incubation, for 6h followed by analysis of Hsp27 phosphorylation on Ser-82 residue

pre-by western blotting As a control, we used a protein phosphatase inhibitor, okadaic acid

As expected from previous results, LY30, alone or in combination with TRAIL, induced a strong phosphorylation of Hsp27 on Ser-82 residue after 6h of treatment (Figure 27A) Interestingly, pre-treatment with okadaic acid significantly enhanced Hsp27 phosphorylation regardless of the treatment the cells were exposed

to, indicating that protein phosphatases, and possibly PP2A, were involved in Hsp27 phosphorylation in our cell system

We next examined a possible effect of LY30 on PP2A activity HeLa cells were treated with LY30, TRAIL or a combination of both for different time PP2A activity was ascertained, in cell lysates depleted of free phosphate, by determining the amount of free phosphate in a reaction by measuring the absorbance of a molybdate:malachite green:phosphate complex Since the buffer used was specific for PP2A and the phosphopeptide (RRA(pT)VA), added as a phosphatase substrate, was specific for Ser/Thr phosphatases, the amount of free phosphate was proportional to

A significant inhibition of PP2A activity could be observed upon treatment with LY30 alone after 1h (Figure 27B) Interestingly, an even greater inhibition was seen after 6h in samples treated with LY30, alone or in combination with TRAIL In addition, although TRAIL treatment alone could inhibit PP2A activity to some extent,

no additive effect could be observed with the combined treatment

In parallel to the study of p38 MAPK and protein phosphatase inhibitors effect

on Hsp27 phosphorylation, we also monitored their effect on cell viability on cells treated with TRAIL for 16h, with or without pre-incubation with LY30 for 1h As seen in Figure 28, inhibition of p38 MAPK by SB203580 led to a significant rescue from cell death in cells exposed to the combined treatment of TRAIL and LY30 Inversely, inhibition of protein phosphatase with okadaic acid resulted in an increase

in cell death for all treatment combination

Taken together, these results indicate that LY30 is able to induce Hsp27 phosphorylation by activating p38 MAKP as well as inhibiting PP2A Moreover, it seems that p38 MAPK activation occurs early and is responsible for the rapid phosphorylation of Hsp27 upon exposure to LY30 The slower inhibition of PP2A phosphatase activity could be responsible for the sustained phosphorylation of Hsp27 observed earlier

Figure 27: Effect of LY30 treatment on PP2A activity

Hela cells were treated for 16h with 20 ng/mL TRAIL with or without pre-incubation with 25 !M LY30 for 1h, in the presence or absence of 5nM okadaic acid Total Hsp27 and phospho-Hsp27 (Ser-82) levels in the whole cell lysates were then assayed

by Western blotting (A) HeLa cells were treated as indicated PP2A activity was then

assayed as described in the Material and Methods section (OA: Okadaic Acid) Data

shown are the mean±S.D of three independent experiments (B) *p<0.01

Figure 28: Effects of p38 MAPK and PP2A inhibitors on cell viability

Hela cells were treated for 16h with 20 ng/mL TRAIL with or without pre-incubation with 25 !M LY30 for 1h, in the presence or absence of 10 !M SB205380 or 5 nM okadaic acid Percentage of cell survival was determined by crystal violet assay Data shown are the mean±S.D of three independent experiments *p<0.01

LY30 disrupts the equilibrium between small size and large size Hsp27 oligomers

Following the confirmation that LY30 was able to modulate Hsp27 phosphorylation in our cell system, we set out to investigate the subsequent effect on Hsp27 oligomerization

To that end, HeLa cells were treated with TRAIL, LY30 or a combination of both for different times Cell extracts were fractionated by ultracentrifugation on glycerol gradient, and each fraction was analyzed for the presence of Hsp27 by slot blotting followed by immunoblotting

In untreated cells, Hsp27 sedimented as complexes of heterogeneous sizes distributed between the top of the gradient (monomers, dimmers and low molecular size oligomers) and fractions corresponding to a high molecular mass (Figure 29A and B) Interestingly, treatment with LY30 alone for 1h lead to a drastic change in the size distribution of Hsp27 oligomers, with a shift towards the lower molecular size oligomers (Figure 29A) Longer treatment with LY30, alone or in combination with TRAIL, lead to a similar shift towards small oligomers (Figure 29B) Likewise, treatment with okadaic acid, which is able to increase Hsp27 phosphorylation as seen earlier, lead to a comparable shift (Figure 29A) Inversely, pre-treatment with the p38 MAPK inhibitor SB203580 abrogated LY30-mediated shift in Hsp27 oligomeric size (Figure 29A)

These results suggest that modulation of Hsp27 phosphorylation by LY30 directly influences the equilibrium between high and low molecular size Hsp27 oligomers, in favor of the latter

Figure 29: Effect of LY30 treatment on Hsp27 oligomers size distribution

HeLa cells were left untreated, treated with 25 !M LY30 for 1h, LY30 for 1h after 2h

pre-incubation with SB203580 or okadaic acid alone for 4h (A) or treated for 6h with

20 ng/mL with or without pre-incubation with 25 !M LY30 for 1h (B) The cell

LY30 affects Hsp27 molecular chaperone activity in vivo

The capacity of Hsp27 to protect proteins from denaturation is proposed to be

in part responsible for its anti-apoptotic ability Hsp27 chaperone activity is known to

be carried out by the fraction of Hsp27 oligomers displaying a high molecular size Given the previous results on Hsp27 oligomerization, we set out to test whether LY30 also had an effect on Hsp27 chaperone activity

HeLa cells transiently expressing the renilla luciferase gene were treated as described in the experimental setup outlined in Figure 30A The chaperone activity was calculated by dividing the luciferase luminescence from the heat-shocked cells by the one from the non-heat-shocked cells Since this assay measures the overall chaperone activity in the cell (i.e the activity of all chaperone proteins), both specific over-expression and silencing of Hsp27 were used to determine the contribution of Hsp27 in the overall chaperone activity (Figure 30B)

As seen in Figure 30C, the heat shock was sufficient to denature luciferase efficiently (HSR0 +/- LY) and a 3h recovery period was enough to observe a significant renaturation of the protein Interestingly, Hsp27 over-expression was able

to enhance protein renaturation by about 140%, whereas Hsp27 silencing reduced it

by 40% as compared to the control, indicating that Hsp27 is responsible for a significant part of the chaperone activity in the cell More importantly, it can be observed that LY30 treatment significantly reduces the chaperone activity Also, when cells over-expressing Hsp27 are considered, this reduction in chaperone activity

is further reduced by LY30, indicating that LY30 specifically targets Hsp27 chaperone activity

Figure 30: Effect of LY30 treatment on in vivo chaperone activity

Experimental setup for the in vivo chaperone experiments Cycloheximide was added

to the cells before treatment to avoid translation of additional luciferase and endogenous chaperones during the heat-shock Chaperone activity was calculated by dividing the renilla luciferase activity from each condition to the respective non-heat

Modulation of Hsp27 expression can modulate HeLa cells viability upon treatment with TRAIL, alone or in combination with LY30

Although we showed that LY30 was able to induce profound biochemical changes of Hsp27 protein, no change in Hsp27 expression could be observed upon treatment with LY30, alone or in combination with TRAIL Thus, we were interested

to see whether artificial modulation of Hsp27 protein level could have an effect on the viability HeLa cells treated with TRAIL and LY30

HeLa cells were transiently transfected with a plasmid permitting the expression of Hsp27 or with siRNAs specifically targeting Hsp27 (Figure 31A) In accordance with previous results, cells pre-incubated with different doses of LY30 showed a decrease in cell viability upon treatment with TRAIL for 16h, the intensity

over-of which was dependent on the dose over-of TRAIL used (Figure 31B) As seen in Figure 31C, over-expression of Hsp27 lessened this decrease in cell viability by up to 20% in cells treated with doses of TRAIL equal or superior to 20ng/mL Inversely, knock-down of Hsp27 expression by siRNAs sensitized cells exposed to the combined treatment, with a 20% difference for all doses of TRAIL used as compared to untransfected cells (Figure 31D) As seen in the histogram summarizing the data for the concentrations of drugs commonly used in other experiments (i.e 25µM LY30 and 20ng/mL TRAIL), Hsp27 over-expression offered a 20% protection, whereas Hsp27 silencing enhanced cell death by 20% Surprisingly, silencing Hsp27 did not sensitized HeLa when TRAIL was used alone, even at the highest dose (Figure 31E)

These results suggest that Hsp27 plays a role in LY30-mediated sensitization

of HeLa cells to TRAIL-induced apoptosis However, Hsp27 does not seem to be

Figure 31: Effect of Hsp27 expression on LY30 sensitization to TRAIL-mediated apoptosis

Hela cells were transiently transfected with either a plasmid driving Hsp27

over-expression or siRNAs specifically targeting Hsp27 mRNA (A) These cells were

exposed to TRAIL (0-250 ng/mL) for 16h in the presence or absence of 1h incubation with various dose of LY30 (0-25 !M) Percentage of cell survival in

pre-control cells (B), Hsp27 over-expressing cells (C) and cells with silenced Hsp27 expression (D), was determined by crystal violet assay Summary histogram for treatment with 25 !M LY30 and 20 ng/mL TRAIL (E) Data shown are the

mean±S.D of three independent experiments *p<0.01