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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: This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting.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.

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

Specificity of test shRNA towards HPV-16 enhancer. a Time dependent fall in the expression of HPV-16 E6 and E7 by NFκBEn–Pr+2-HPV-16–E6/E7, in SiHa cells, showed maximum suppression after 5 days (p < 0.05 at all-time points). The apparent increase in E6 and E7 mRNA on the 6th day compared with 5th day was statistically insignificant (p = 0.22). b, c Decrease in E6 and E7 mRNA levels is seen in both HPV-16 positive cell lines SiHa and CaSki and the fall in E6/E7 expression is in concordance with strength of the construct driving shRNA expression. NFκBEn–Pr+2-HPV-16–E6/E7 significantly decreased HPV-16 E6/E7 mRNA levels over PLAPPr+2-HPV-16–E6/E7 in SiHa cell line (p = 0.022 and p = 0.030 for E6 and E7, respectively) and CaSki (p = 0.041 and p = 0.017 for E6 and E7, respectively). d, e Post HPV-16 E6/E7 suppression by shRNA, significant increase in the expression of p53 target genes was observed in SiHa and CaSki cells at the mRNA level. f and g Restoration of p53 protein, post HPV-16 E6/E7 suppression corroborated with the mRNA levels of PUMA and NOXA. h and i Decrease in the HPV-16 E7 expression, post shRNA treatment, significantly reduced levels of E2FI candidate genes like cyclin A2 and E (p < 0.05).
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Fig2: Specificity of test shRNA towards HPV-16 enhancer. a Time dependent fall in the expression of HPV-16 E6 and E7 by NFκBEn–Pr+2-HPV-16–E6/E7, in SiHa cells, showed maximum suppression after 5 days (p < 0.05 at all-time points). The apparent increase in E6 and E7 mRNA on the 6th day compared with 5th day was statistically insignificant (p = 0.22). b, c Decrease in E6 and E7 mRNA levels is seen in both HPV-16 positive cell lines SiHa and CaSki and the fall in E6/E7 expression is in concordance with strength of the construct driving shRNA expression. NFκBEn–Pr+2-HPV-16–E6/E7 significantly decreased HPV-16 E6/E7 mRNA levels over PLAPPr+2-HPV-16–E6/E7 in SiHa cell line (p = 0.022 and p = 0.030 for E6 and E7, respectively) and CaSki (p = 0.041 and p = 0.017 for E6 and E7, respectively). d, e Post HPV-16 E6/E7 suppression by shRNA, significant increase in the expression of p53 target genes was observed in SiHa and CaSki cells at the mRNA level. f and g Restoration of p53 protein, post HPV-16 E6/E7 suppression corroborated with the mRNA levels of PUMA and NOXA. h and i Decrease in the HPV-16 E7 expression, post shRNA treatment, significantly reduced levels of E2FI candidate genes like cyclin A2 and E (p < 0.05).

Mentions: NFκBEn–Pr+2-HPV-16–E6/E7 or NFκBEn–Pr+2-HPV-16–E6/E7 Scr were transfected in SiHa cells and fall in expression of HPV-16 E6 and E7 was evaluated consecutively for 6 days This decrease was significant at all-time points (p < 0.05) and was maximum on the 5th day (Fig. 2a). Slight apparent increase on the 6th day compared to the 5th day was insignificant (p = 0.22). Fall in the HPV-16 E6 and E7 expression by other shRNA constructs in SiHa cells was also significant (Fig. 2b; p < 0.05). Similar trend was observed in CaSki cells (Fig. 2c). No significant decrease was observed in HeLa cells (p > 0.05; Additional file 4: Figure S4A) illustrating the specificity of the shRNA for HPV-16. Further, the potential to knockdown HPV-16 E6 and E7 expression by tissue specific NFκBEn–Pr+2-HPV-16–E6/E7 was comparable to tissue non-specific CMVPr–HPV-16–E6/E7 (p > 0.05). However, our NFκB–PLAP promoter, unlike CMV promoter, was active only under neoplastic condition. The activity of NFκBEn–Pr+2-HPV-16–E6/E7 was significantly higher than PLAPPr+2-HPV-16–E6/E7 in both SiHa and CaSki cells (p < 0.05). Hence, we were able to increase the transcriptional activation of the downstream TGS inducing shRNA, while retaining its tumour selective expression by fusing four copies of NFκB responsive element upstream to the PLAP promoter.Fig. 2


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)

Specificity of test shRNA towards HPV-16 enhancer. a Time dependent fall in the expression of HPV-16 E6 and E7 by NFκBEn–Pr+2-HPV-16–E6/E7, in SiHa cells, showed maximum suppression after 5 days (p < 0.05 at all-time points). The apparent increase in E6 and E7 mRNA on the 6th day compared with 5th day was statistically insignificant (p = 0.22). b, c Decrease in E6 and E7 mRNA levels is seen in both HPV-16 positive cell lines SiHa and CaSki and the fall in E6/E7 expression is in concordance with strength of the construct driving shRNA expression. NFκBEn–Pr+2-HPV-16–E6/E7 significantly decreased HPV-16 E6/E7 mRNA levels over PLAPPr+2-HPV-16–E6/E7 in SiHa cell line (p = 0.022 and p = 0.030 for E6 and E7, respectively) and CaSki (p = 0.041 and p = 0.017 for E6 and E7, respectively). d, e Post HPV-16 E6/E7 suppression by shRNA, significant increase in the expression of p53 target genes was observed in SiHa and CaSki cells at the mRNA level. f and g Restoration of p53 protein, post HPV-16 E6/E7 suppression corroborated with the mRNA levels of PUMA and NOXA. h and i Decrease in the HPV-16 E7 expression, post shRNA treatment, significantly reduced levels of E2FI candidate genes like cyclin A2 and E (p < 0.05).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
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Fig2: Specificity of test shRNA towards HPV-16 enhancer. a Time dependent fall in the expression of HPV-16 E6 and E7 by NFκBEn–Pr+2-HPV-16–E6/E7, in SiHa cells, showed maximum suppression after 5 days (p < 0.05 at all-time points). The apparent increase in E6 and E7 mRNA on the 6th day compared with 5th day was statistically insignificant (p = 0.22). b, c Decrease in E6 and E7 mRNA levels is seen in both HPV-16 positive cell lines SiHa and CaSki and the fall in E6/E7 expression is in concordance with strength of the construct driving shRNA expression. NFκBEn–Pr+2-HPV-16–E6/E7 significantly decreased HPV-16 E6/E7 mRNA levels over PLAPPr+2-HPV-16–E6/E7 in SiHa cell line (p = 0.022 and p = 0.030 for E6 and E7, respectively) and CaSki (p = 0.041 and p = 0.017 for E6 and E7, respectively). d, e Post HPV-16 E6/E7 suppression by shRNA, significant increase in the expression of p53 target genes was observed in SiHa and CaSki cells at the mRNA level. f and g Restoration of p53 protein, post HPV-16 E6/E7 suppression corroborated with the mRNA levels of PUMA and NOXA. h and i Decrease in the HPV-16 E7 expression, post shRNA treatment, significantly reduced levels of E2FI candidate genes like cyclin A2 and E (p < 0.05).
Mentions: NFκBEn–Pr+2-HPV-16–E6/E7 or NFκBEn–Pr+2-HPV-16–E6/E7 Scr were transfected in SiHa cells and fall in expression of HPV-16 E6 and E7 was evaluated consecutively for 6 days This decrease was significant at all-time points (p < 0.05) and was maximum on the 5th day (Fig. 2a). Slight apparent increase on the 6th day compared to the 5th day was insignificant (p = 0.22). Fall in the HPV-16 E6 and E7 expression by other shRNA constructs in SiHa cells was also significant (Fig. 2b; p < 0.05). Similar trend was observed in CaSki cells (Fig. 2c). No significant decrease was observed in HeLa cells (p > 0.05; Additional file 4: Figure S4A) illustrating the specificity of the shRNA for HPV-16. Further, the potential to knockdown HPV-16 E6 and E7 expression by tissue specific NFκBEn–Pr+2-HPV-16–E6/E7 was comparable to tissue non-specific CMVPr–HPV-16–E6/E7 (p > 0.05). However, our NFκB–PLAP promoter, unlike CMV promoter, was active only under neoplastic condition. The activity of NFκBEn–Pr+2-HPV-16–E6/E7 was significantly higher than PLAPPr+2-HPV-16–E6/E7 in both SiHa and CaSki cells (p < 0.05). Hence, we were able to increase the transcriptional activation of the downstream TGS inducing shRNA, while retaining its tumour selective expression by fusing four copies of NFκB responsive element upstream to the PLAP promoter.Fig. 2

Bottom Line: This was packaged in a Sendai virus envelope incorporating a single chain variable fragment antibody (scFv) for antibody mediated targeting.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.

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