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A novel placental like alkaline phosphatase promoter driven transcriptional silencing combined with single chain variable fragment antibody based virosomal delivery for neoplastic cell targeting [corrected].

Khan I, Zakaria MK, Kumar M, Mani P, Chattopadhyay P, Sarkar DP, Sinha S - J Transl Med (2015)

Bottom Line: Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties.There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes.Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, 110029, India. imranaiims@yahoo.com.

ABSTRACT

Background: Placental like alkaline phosphatase (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues [corrected]. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting.

Methods: PLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties.

Results: Reduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region.

Conclusion: A combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.

No MeSH data available.


Related in: MedlinePlus

Caspase activity and mechanism involved in TGS. a HPV-16 E6/E7 suppression by NFκBEn–Pr+2-HPV-16–E6/E7, 5 days post virosomal delivery, led to increase in the caspase 3/7 activity in SiHa (p < 0.005) and CaSki (p = 0.02) cell lines. No such increase was observed in HeLa (p = 0.38) and CHO cells (p = 0.41). b Chip assay in SiHa cells transfected with NFκBEn–Pr+2 HPV-16–E6/E7 showed the silencing of the target region as a result of heterochromatization by methylation of histone tails (H3K9Me2 and H3K27Me3; p < 0.001 for both). However, cells pre-treated with TSA, did not show significant enrichment indicating that in the presence of TSA, shRNA failed to induce significant heterochromatization (p > 0.05). c and d No difference in the methylation pattern of the CpG islands, around the target LCR region, of SiHa cell line was observed by bisulphite PCR and followed by DNA sequencing. e The levels of enhancer associated transcripts decreased significantly post chimeric scFv-F virosomal delivery of NFκBEn–Pr+2 HPV-16–E6/E7 construct in both HPV-16 integrated SiHa and CaSki cells (p < 0.05).
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Fig5: Caspase activity and mechanism involved in TGS. a HPV-16 E6/E7 suppression by NFκBEn–Pr+2-HPV-16–E6/E7, 5 days post virosomal delivery, led to increase in the caspase 3/7 activity in SiHa (p < 0.005) and CaSki (p = 0.02) cell lines. No such increase was observed in HeLa (p = 0.38) and CHO cells (p = 0.41). b Chip assay in SiHa cells transfected with NFκBEn–Pr+2 HPV-16–E6/E7 showed the silencing of the target region as a result of heterochromatization by methylation of histone tails (H3K9Me2 and H3K27Me3; p < 0.001 for both). However, cells pre-treated with TSA, did not show significant enrichment indicating that in the presence of TSA, shRNA failed to induce significant heterochromatization (p > 0.05). c and d No difference in the methylation pattern of the CpG islands, around the target LCR region, of SiHa cell line was observed by bisulphite PCR and followed by DNA sequencing. e The levels of enhancer associated transcripts decreased significantly post chimeric scFv-F virosomal delivery of NFκBEn–Pr+2 HPV-16–E6/E7 construct in both HPV-16 integrated SiHa and CaSki cells (p < 0.05).

Mentions: Significant increase in caspase 3/7 activity was observed in SiHa and CaSki cells five days post virosomal delivery of NFκBEn+2-HPV-16–E6/E7. However, no such increase was seen in HeLa and CHO cells (Fig. 5a).Fig. 5


A novel placental like alkaline phosphatase promoter driven transcriptional silencing combined with single chain variable fragment antibody based virosomal delivery for neoplastic cell targeting [corrected].

Khan I, Zakaria MK, Kumar M, Mani P, Chattopadhyay P, Sarkar DP, Sinha S - J Transl Med (2015)

Caspase activity and mechanism involved in TGS. a HPV-16 E6/E7 suppression by NFκBEn–Pr+2-HPV-16–E6/E7, 5 days post virosomal delivery, led to increase in the caspase 3/7 activity in SiHa (p < 0.005) and CaSki (p = 0.02) cell lines. No such increase was observed in HeLa (p = 0.38) and CHO cells (p = 0.41). b Chip assay in SiHa cells transfected with NFκBEn–Pr+2 HPV-16–E6/E7 showed the silencing of the target region as a result of heterochromatization by methylation of histone tails (H3K9Me2 and H3K27Me3; p < 0.001 for both). However, cells pre-treated with TSA, did not show significant enrichment indicating that in the presence of TSA, shRNA failed to induce significant heterochromatization (p > 0.05). c and d No difference in the methylation pattern of the CpG islands, around the target LCR region, of SiHa cell line was observed by bisulphite PCR and followed by DNA sequencing. e The levels of enhancer associated transcripts decreased significantly post chimeric scFv-F virosomal delivery of NFκBEn–Pr+2 HPV-16–E6/E7 construct in both HPV-16 integrated SiHa and CaSki cells (p < 0.05).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4524171&req=5

Fig5: Caspase activity and mechanism involved in TGS. a HPV-16 E6/E7 suppression by NFκBEn–Pr+2-HPV-16–E6/E7, 5 days post virosomal delivery, led to increase in the caspase 3/7 activity in SiHa (p < 0.005) and CaSki (p = 0.02) cell lines. No such increase was observed in HeLa (p = 0.38) and CHO cells (p = 0.41). b Chip assay in SiHa cells transfected with NFκBEn–Pr+2 HPV-16–E6/E7 showed the silencing of the target region as a result of heterochromatization by methylation of histone tails (H3K9Me2 and H3K27Me3; p < 0.001 for both). However, cells pre-treated with TSA, did not show significant enrichment indicating that in the presence of TSA, shRNA failed to induce significant heterochromatization (p > 0.05). c and d No difference in the methylation pattern of the CpG islands, around the target LCR region, of SiHa cell line was observed by bisulphite PCR and followed by DNA sequencing. e The levels of enhancer associated transcripts decreased significantly post chimeric scFv-F virosomal delivery of NFκBEn–Pr+2 HPV-16–E6/E7 construct in both HPV-16 integrated SiHa and CaSki cells (p < 0.05).
Mentions: Significant increase in caspase 3/7 activity was observed in SiHa and CaSki cells five days post virosomal delivery of NFκBEn+2-HPV-16–E6/E7. However, no such increase was seen in HeLa and CHO cells (Fig. 5a).Fig. 5

Bottom Line: Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties.There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes.Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, 110029, India. imranaiims@yahoo.com.

ABSTRACT

Background: Placental like alkaline phosphatase (PLAP), an oncofetal antigen, is highly expressed in germ cell, cervical, ovarian and several other tumour types but minimally in normal tissues [corrected]. The expression of a PLAP promoter based transcriptional unit following antigen mediated cell specific delivery is a possible approach for tumour targeting.

Methods: PLAP promoter alone or in combination with NFκB DNA response elements was used for expressing shRNA targeting the long control region (LCR) of human papillomavirus (HPV)-16 oncogenes E6 and E7 via transcriptional gene silencing in PLAP expressing cervical cancer cell lines, SiHa and CaSki. This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting. Specificity and efficacy of the shRNA was assessed by studying the heterochromatization, down regulation of the HPV-16 E6/E7 genes and subsequent effects on their targets and cell growth properties.

Results: Reduction of HPV-16 E6 and E7 expression by TGS led to the activation of the previously suppressed target genes of p53 (PUMA and NOXA) and Rb (cyclins A2 and E). Cell death was seen only in PLAP expressing HPV-16 infected SiHa and CaSki cells but not in the HPV-18 integrated HeLa and non-PLAP CHO cells. There was reduction in the enhancer associated transcripts of the long control region (LCR) of HPV-16 E6/E7 genes. Also, an increase in the enrichment of dimethylated histone three lysine nine (H3K9Me2) and trimethylated histone three lysine twenty-seven (H3K27Me3) was observed by ChIP assay, which decreased upon trichostatin A treatment, indicating a possible mechanism for the heterochromatization of the target LCR region.

Conclusion: A combination of novel PLAP promoter and antibody based specificities has the potential for being developed as a possible therapeutic strategy for PLAP positive neoplasia.

No MeSH data available.


Related in: MedlinePlus