PROTOCOLO REUNIDOS

UI ztcof1 MO p53 MO + p53 MO

ztcof1 MO

21 hpf

21 hpf

TUNEL

The best attempts to rescue the phenotype of TCS were performed by inhibiting tumor suppressor p53 in Tcof1 heterozygous mice and in zebrafish by co-injecting p53 morpholinos along with that of ztcof. Thus, interrupting the apoptosis pathway of TCS proved to be sufficient in preventing the onset of the disease in these two animal models. However, the loss of p53 in mice resulted in tumor formation and this limits the potential of using this rescue in preventing onset TCS in humans. Therefore, it is important to identify other members of the apoptotic pathway which if lost, will not cause cancer.

P21 is a very strong candidate for many reasons described earlier in chapter 2. In addition, p21 homozygous knockout mice do not develop tumors (Deng C et al., 1995) which overcome the obstacle of tumor formation when p53 is knocked out. The pro- apoptotic protein, Bnip3l, is another strong candidate that appeared to be upregulated in TCOF1 knockdown mouse neuroblastoma cell line (Mogass et al., 2004) and may be down-stream of p53 in the apoptosis pathway. The cell cycle arrest in G1 phase has been observed in TCS models (Dixon et al., 2008; Lin Li et al., 2006), which makes p27 a potential member of the pathway. Therefore, the level of expression of these three genes was examined by in-situ hybridization, in ztcof1 morpholino injected embryos.

Materials and Methods

Adult fish were maintained at approximately 28˚C on a 14 hour/10 hour light/dark cycle. Wild type strains (AB strain or AB/TU) were used for all experiments unless otherwise indicated. Embryos were incubated at 28˚C until desired stage was reached. Probe Preparation

Approximately 5 µg of the following plasmids were linearized using the restriction enzyme EcoRI and approximately 1 µg served as templates for transcription reaction using the indicated RNA polymerase: pBS-IISK-p27 – T7 RNA polymerase, pBluescript SK- Bnip3l – T7 RNA polymerase. P21 which was purchased in a pSPORT1 vector (ZFIN) did not contain an RNA polymerase promoter. Therefore, the p21 plasmid was digested with EcoRI and the cDNA was amplified with PCR by a T3 promoter coded in the 3’ primer. The digested plasmids and PCR product were purified for the transcription reaction with GeneClean purification kit (Qbiogene). Probes were then diluted 1:100 to 1:200 in [50% formamide, 5x SSC, 50 mg/ml heparin, 500 mg/ml tRNA, 0.1% Tween 20, 9 mM citric acid].

In situ Protocol

Embryos staged as described by Kimmel et al. (1993) were fixed 24, 48 and 72 hours in 4% paraformaldehyde in 1XPBS (phosphate-buffered saline), hand dechorionated and dehydrated overnight in methanol at -20°C. Then the embryos were rehydrated stepwise in methanol/PBT and finally put back in 100% PBT (1´ PBS 0.1% Tween 20). Embryos older than 24 hours were treated with proteinase K (20 mg/ml in PBT) 1 minute at room temperature for every hour of embryo age. Prpteinase K was aspirated and embryos were post-fixed with 4% PFA in 1xPBS for 20 minutes and then washed several times with 1xPBT 5 times for 5 minutes each. The embryos were prehybridized at least 1

hour at 65°C in hybridization buffer [50% formamide, 5x SSC, 50 mg/ml heparin, 500 mg/ ml tRNA, 0.1% Tween 20, 9 mM citric acid]. The hybridization was done in the same buffer containing 50 ng to 100 ng of probe overnight at 70°C. Then the embryos were washed at 65°C for 10 minutes in [75% hybridization buffer, 25% 2x SSC], 10 minutes in [50% hybridization buffer, 50% 2x SSC], 10 minutes in [25% hybridization mix, 75% 2xSSC], 10 minutes in 2x SSC, 2 times 20 minutes in 0.2x SSC. Further washes were performed at room temperature for 5 minutes in [75% 0.2x SSC, 25% PBT], 5 minutes in [50% 0.2x SSC, 50% PBT], 5 minutes in [25% 0.2x SSC, 75% PBT], 5 minutes in PBT, and then 1 hour in [PBT with 2 mg/ml BSA (bovine serum albumin), 2% sheep serum]. Then the embryos were incubated overnight at 4°C with the preabsorbed alkalinephosphatase- coupled anti-digoxigenin antiserum (described in Boehringer) at a 1/10,000 dilution in a PBT buffer containing 2 mg/ml BSA, 2% sheep serum. Finally the embryos were washed 6 times for 15 minutes each in PBT at room temperature. Detection was performed in alkaline phosphatase reaction buffer described in the Boehringer. When the color was developed, the reaction was stopped in 1x PBT and embryos were dehydrated and cleared in MeOH overnight. The embryos were then rehydrated in MeOH/PBT series and equilibrated in 50% glycerol in 1 X PBS for viewing and photographing.

Morpholino

Ztcof1 morpholino that was used in this study has been previously described (Holser, 2007).

Results

Examining the levels of expression of the p27 gene in zebrafish embryos that were injected with ztcof1 MO showed no difference from the ones that were injected with the

Mismatch (MM) (Figure 11). However, p21, which is one of the major transcriptional targets of p53, and is known to be responsible for causing G1 phase cell cycle arrest in response to a variety of stimuli was found to be upregulated in ztcof1 MO injected embryos compared to MM injected ones. This was observed in the 24 (data not shown), 48 and 72 hpf (figure 12). Bnip3l, which is also a transcriptional target of p53, was not found to be upregulated in ztcof1 MO injected embryos as compared to the embryos injected with the MM (figure 13).

Discussion

p27 is known to bind to cyclin D either alone, or when complexed to its catalytic subunit CDK4. In doing so, p27 inhibits the catalytic activity of Cdk4, which means that it prevents Cdk4 from adding phosphate residues to its principal substrate, the retinoblastoma (pRb) protein. This inhibits the dissociation of E2F-1, which is a transcription factor, from pRb and hence from mediating the trans-activation of E2F-1 target genes that facilitate the G1/S transition. Therefore, increased levels of the p27protein typically cause cells to arrest in the G1 phase of the cell cycle. However, the in-situ hybridization showed no significant increase in p27 in MO injected embryos when compared to one injected with the MM. This indicates that, it could be another G1/S checkpoint that causes the G1 phase cell cycle arrest observed in TCS.

The CDK inhibitor p21 was upregulated in embryos that were injected with ztcof1 MO compared to embryos injected with the MM. This result is expected because p53 is stabilized when ztcof1 is knocked down and as was mentioned previously, p53 promotes transcriptional activation of p21. The exact role of p21 in apoptosis remains unclear, but the G1 phase cell cycle arrest must be a very important part of it. It would be interesting to

investigate the potential of rescuing the onset of TCS in any of the models by inhibiting the expression of p21.

Bnip3l, which is a proapoptotic protein that has a p53 binding sequence in it’s gene, was not found to be upregulated in embryos injected with the MO, compared to the ones injected with the MM. Therefore, it is more likely that the apoptosis does not involve Bnip3l.

Future work

In summary, when Tcof1 is reduced, ribosome biogenesis is affected and this leads to the activation of p53 through its stabilization. In turn, p53 promotes G1 phase cell cycle arrest and ultimately apoptosis. The inhibition of this apoptosis pathway, by inhibiting p53 expression, proved to be sufficient in preventing the onset of TCS in the mouse model (Dixon et al., 2008). The fact that most mice in this rescue developed tumors within 3 to 6 months limits the potential of using it when treating TCS in humans. Therefore, further studies should be performed to find other members in this pathway and try to rescue the phenotype using this knowledge.

When ribosome biogenesis is impaired, L11 diffuse into the nucleoplasm where it sequesters HDM2, ultimately stabilizing p53 (Lohrum et al., 2003). This could be occurring upstream of p53-mediated cell cycle arrest and apoptosis seen in TCS. A rescue experiment could be performed by the inhibition of the expression of L11. This should be first investigated using the zebrafish model, by the co-injection of ztcof1 MO and L11 MO followed by TUNEL assay to test if the knockdown of L11 would prevent the neuroepithelial apoptosis and rescue phenotype.

the knock down of ztcof1 in zebrafish embryos, the possibility of preventing the onset of TCS by blocking p21 is raised. The most informative and suitable model to use for this rescue is TCS mouse model obtained by breeding DBA Tcof1+/- _{mice and C57BL/6 wild}

type mice to generate mixed DBA x C57BL/6 F1 progeny (Dixon, J et al., 2006). F1

progeny of DBA Tcof1+/- _{mice bred with C57BL/6 p21} +/- _{to generate compound Tcof1}+/-

p21 +/- _{mice, will be compared to DBA Tcof1}+/- _{mice bred with C57BL/6 wild type mice as}

controls. Morphology of the head can then be assessed. In addition, to evaluate the efficacy of p21 inhibition in preventing the pathogenesis of TCS, the segregated streams of migrating cranial neural crest cells can be examined using in-situ hybridization detecting Sox10 (at E9.0) which is a cranial neural crest marker (reviewed in Kelsh RN et al., 2006). If inhibition of p21 should prevent the pathogenesis of TCS, in-situ hybridization would show the discrete segregated migrating streams of neural crest cells resembling wild type. Another rescue experiment can be performed using the zebrafish model by the co-injection of ztcof1 and p21 morpholinos followed by TUNEL assay to test if the knockdown of p21 will prevent apoptosis.

The microarray study (Mogass et al., 2004) revealed a number of genes that were found to be positively or negatively correlated with TCOF1 expression. These results should be confirmed with RT-PCR which would also reveal the extent of that correlation. Strongly correlated genes should then be investigated according to their functional classification, as either cell cycle genes, metabolism genes... etc.

For example, Rasa1, a known cell cycle protein, was found to be coordinately regulated with Tcof1 expression. Rasa1 is known to reduce p38 MapK activity through the promotion of GDP-Ras so less phosphate is available for phosphorylation. P38 MapK is a

member of mitogen-activated protein kinases and is known to activate p53 by directly phosphorylating the protein. Therefore, there is a possibility that L11 protein is promoting p53 stabilization while increased levels of p38 MapK is promoting activation of the protein by directly phophorylating it, when Tcof1 is knocked down. The levels of p38 Mapk protein should be tested by Western Blot in the TCOF1 knocked down neuroblastoma cell line. Due to decreased levels of Rasa1, it is expected that protein levels of p38 Mapk be increased in the TCOF1 knocked down cell line.

If levels of p38 MapK were found to be elevated, it would be interesting to investigate the potential of inhibiting the expression of this protein to prevent apoptosis. Although previous experiments to inhibit the neuroepithelial apoptosis by knocking out p53 resulted in tumors, inhibiting the phosphorylation of p53 by p38 MapK would not affect phophorylation mediated by kinases implicated in the genome integrity checkpoints. This would overcome the obstacle of tumor formation when p53 in knocked out. Another protein called fatty acid binding protein-3 (Fabp3) was found to be negatively correlated with Tcof1 expression. Fabp3 is also called mammary derived growth inhibitor (MDGI) due to its potential role as an anti-tumor protein. Due to abnormally increased levels of this protein when TCOF1 is knocked down, it is possible that it may be mediating apoptosis seen in TCS. Therefore, it would be interesting to inhibit the expression of this protein in the TCOF1 KD neuroblastoma cell line using siRNA, and then assess apoptosis by TUNEL assay and see if the inhibition reduces apoptosis.

Figure 11: p27 expression was found to be the same in the MO injected embryos and MM in the 48 and 72 hpf. a and c show MM injected embryos at 48 and 72 hpf respectively. b and d show MO injected embryos at 48 and 72 hpf respectively.

a b

c d

Figure 12: The level of expression of p21 was found to be increased in embryos that were injected with the MO compared to the ones injected with the MM at both the 48 and 72 hpf stages. a and c show MM injected embryos at 48 and 72 hpf respectively. b and d show MO injected embryos at 48 and 72 hpf respectively

a b

c d

Figure 13: Bnip3l expression was found to be the same in the MO injected embryos and MM in the 48 and 72 hpf. a and c show MM injected embryos at 48 and 72 hpf

a b

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