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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

Kinetics of chimeric scFv-F-virosome fusion and knockdown effects post virosomal delivery. a Fusion of R18 labelled chimeric Sendai F-virosomes was determined by fluorescence dequenching assay; significant fusion was observed only in PLAP positive cells but not in non-PLAP CHO cells. scFv virosomes with inactivated F-protein (HC: heat control) displayed poor fusion with HeLa cells confirming fusion specificity via scFv. b Time dependant fall in expression of HPV-16 E6/E7 by NFκBEn–Pr+2-HPV-16–E6/E7, post chimeric virosomal delivery, was comparable with conventional transfection results. c–f Decrease in the expression of HPV-16 E6/E7 and increase in the expression of p53 target genes was observed in both SiHa and CaSki cells and it was in accordance with strength of the shRNA construct. g Amelioration in p53 in SiHa and CaSki, at the protein level, followed the same trend. h and i Post scFv F-virosomal delivery of the shRNA constructs, significant decrease in the expression of E2FI candidate genes (cyclin A2 and E) was observed in SiHa and CaSki cell lines (p < 0.05).
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Fig4: Kinetics of chimeric scFv-F-virosome fusion and knockdown effects post virosomal delivery. a Fusion of R18 labelled chimeric Sendai F-virosomes was determined by fluorescence dequenching assay; significant fusion was observed only in PLAP positive cells but not in non-PLAP CHO cells. scFv virosomes with inactivated F-protein (HC: heat control) displayed poor fusion with HeLa cells confirming fusion specificity via scFv. b Time dependant fall in expression of HPV-16 E6/E7 by NFκBEn–Pr+2-HPV-16–E6/E7, post chimeric virosomal delivery, was comparable with conventional transfection results. c–f Decrease in the expression of HPV-16 E6/E7 and increase in the expression of p53 target genes was observed in both SiHa and CaSki cells and it was in accordance with strength of the shRNA construct. g Amelioration in p53 in SiHa and CaSki, at the protein level, followed the same trend. h and i Post scFv F-virosomal delivery of the shRNA constructs, significant decrease in the expression of E2FI candidate genes (cyclin A2 and E) was observed in SiHa and CaSki cell lines (p < 0.05).

Mentions: Real time fusion kinetics by fluorescence dequenching assay showed that the chimeric scFv-F virosomes specifically fused with PLAP positive cell lines (HeLa, CaSki and SiHa) but not with non PLAP cell line CHO which does not express PLAP. Inactivated chimeric virosomes (HC: Heat control), displayed negligible fusion with HeLa cells (Fig. 4a). The difference in the fusion observed might be dependent upon the number of PLAP molecules expressed by various cell types. Luciferase expression constructs (NFκBEn–Pr+24-luc; PLAPPr+24-luc and SV40-luc) packaged and delivered by chimeric scFv-F virosomes showed significant activity only in PLAP positive cells (Additional file 5: Figure S5A). It differed from lipofectamine based transfections as tissue non-specific SV40-luc did not elicit appreciable activity in non-PLAP cells (Fig. 1d, e). Time dependent fall in HPV-16 E6 and E7 levels, post chimeric virosomal delivery, in SiHa cells (Fig. 4b) were comparable to that by conventional methods (Fig. 2a). Significant fall in the expression of HPV-16 E6 and E7 mRNA was seen both in SiHa and CaSki cells (p < 0.05 for both; Fig. 4c, d). TGS was not effective in HeLa cells due to specificity of shRNA towards HPV-16 (Additional file 6: Figure S6A)Fig. 4


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)

Kinetics of chimeric scFv-F-virosome fusion and knockdown effects post virosomal delivery. a Fusion of R18 labelled chimeric Sendai F-virosomes was determined by fluorescence dequenching assay; significant fusion was observed only in PLAP positive cells but not in non-PLAP CHO cells. scFv virosomes with inactivated F-protein (HC: heat control) displayed poor fusion with HeLa cells confirming fusion specificity via scFv. b Time dependant fall in expression of HPV-16 E6/E7 by NFκBEn–Pr+2-HPV-16–E6/E7, post chimeric virosomal delivery, was comparable with conventional transfection results. c–f Decrease in the expression of HPV-16 E6/E7 and increase in the expression of p53 target genes was observed in both SiHa and CaSki cells and it was in accordance with strength of the shRNA construct. g Amelioration in p53 in SiHa and CaSki, at the protein level, followed the same trend. h and i Post scFv F-virosomal delivery of the shRNA constructs, significant decrease in the expression of E2FI candidate genes (cyclin A2 and E) was observed in SiHa and CaSki cell lines (p < 0.05).
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Related In: Results  -  Collection

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Fig4: Kinetics of chimeric scFv-F-virosome fusion and knockdown effects post virosomal delivery. a Fusion of R18 labelled chimeric Sendai F-virosomes was determined by fluorescence dequenching assay; significant fusion was observed only in PLAP positive cells but not in non-PLAP CHO cells. scFv virosomes with inactivated F-protein (HC: heat control) displayed poor fusion with HeLa cells confirming fusion specificity via scFv. b Time dependant fall in expression of HPV-16 E6/E7 by NFκBEn–Pr+2-HPV-16–E6/E7, post chimeric virosomal delivery, was comparable with conventional transfection results. c–f Decrease in the expression of HPV-16 E6/E7 and increase in the expression of p53 target genes was observed in both SiHa and CaSki cells and it was in accordance with strength of the shRNA construct. g Amelioration in p53 in SiHa and CaSki, at the protein level, followed the same trend. h and i Post scFv F-virosomal delivery of the shRNA constructs, significant decrease in the expression of E2FI candidate genes (cyclin A2 and E) was observed in SiHa and CaSki cell lines (p < 0.05).
Mentions: Real time fusion kinetics by fluorescence dequenching assay showed that the chimeric scFv-F virosomes specifically fused with PLAP positive cell lines (HeLa, CaSki and SiHa) but not with non PLAP cell line CHO which does not express PLAP. Inactivated chimeric virosomes (HC: Heat control), displayed negligible fusion with HeLa cells (Fig. 4a). The difference in the fusion observed might be dependent upon the number of PLAP molecules expressed by various cell types. Luciferase expression constructs (NFκBEn–Pr+24-luc; PLAPPr+24-luc and SV40-luc) packaged and delivered by chimeric scFv-F virosomes showed significant activity only in PLAP positive cells (Additional file 5: Figure S5A). It differed from lipofectamine based transfections as tissue non-specific SV40-luc did not elicit appreciable activity in non-PLAP cells (Fig. 1d, e). Time dependent fall in HPV-16 E6 and E7 levels, post chimeric virosomal delivery, in SiHa cells (Fig. 4b) were comparable to that by conventional methods (Fig. 2a). Significant fall in the expression of HPV-16 E6 and E7 mRNA was seen both in SiHa and CaSki cells (p < 0.05 for both; Fig. 4c, d). TGS was not effective in HeLa cells due to specificity of shRNA towards HPV-16 (Additional file 6: Figure S6A)Fig. 4

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