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Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

Shuda M, Guastafierro A, Geng X, Shuda Y, Ostrowski SM, Lukianov S, Jenkins FJ, Honda K, Maricich SM, Moore PS, Chang Y - PLoS ONE (2015)

Bottom Line: To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally.Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation.Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53- setting.

View Article: PubMed Central - PubMed

Affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53- setting.

No MeSH data available.


Related in: MedlinePlus

MCV sT expression is lethal in mice.(A) Design of the ROSA26-CAG-LNL-MCVsTco, a ROSA26 knock-in construct encoding codon-optimized MCV sT with flanking loxP-STOP-Neo-loxP (LNL) sequence, recombined with the ROSA26 genomic locus. ROSA26-CAG-LNL-MCVsTco crossed with UbcCreERT2 or Atoh1CreERT2 mice allows for TMX-induced Cre recombinase excision of the loxP-STOP-Neo-loxP sequence that results in MCV sT expression under the CAG promoter. (B) MCV sT expression is lethal in UbcCreERT2; ROSAsT transgenic mice. Kaplan-Meier curve of high dose (0.2 mg/g) and low dose (0.02 mg/g) TMX injected mice. High dose TMX injection caused rapid weight loss and mice reached the euthanasia criterion (20% loss of body weight) within 5 days of the injection (n = 4, solid orange). Survival of the control UbcCreERT2 mice (n = 7, dotted orange) was not affected by TMX injection. UbcCreERT2; ROSAsT mice injected with lower dose TMX exhibited significantly prolonged survival compared to high dose TMX (n = 18, solid red) and control mice were unaffected (n = 10, dotted red) (C) Multi-tissue MCV sT protein expression in UbcCreERT2; ROSAsT mice that died immediately after high dose TMX injection (< 5 days). Lower dose TMX injection induces less MCV sT protein expression in a mouse that died at day 7 after the injection as detected by immunoblotting using an antibody raised against MCV sT, CM8E6 [22]. MCV sT vector or empty vector transfected HEK293 cells were used as a positive and a negative control, respectively. Equal amounts of sT-transfected HEK293 cell lysates were loaded for normalization across different blots. Hsp70/Hsc70 was detected as a protein quality control. (D) MCV sT protein expression was maintained in tissues of UbcCreERT2; ROSAsT mice that survived over 70 days after low dose TMX injection. Immunoblots were performed as in (C). Tissue lysates from ROSAsT were used as a negative control.
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pone.0142329.g001: MCV sT expression is lethal in mice.(A) Design of the ROSA26-CAG-LNL-MCVsTco, a ROSA26 knock-in construct encoding codon-optimized MCV sT with flanking loxP-STOP-Neo-loxP (LNL) sequence, recombined with the ROSA26 genomic locus. ROSA26-CAG-LNL-MCVsTco crossed with UbcCreERT2 or Atoh1CreERT2 mice allows for TMX-induced Cre recombinase excision of the loxP-STOP-Neo-loxP sequence that results in MCV sT expression under the CAG promoter. (B) MCV sT expression is lethal in UbcCreERT2; ROSAsT transgenic mice. Kaplan-Meier curve of high dose (0.2 mg/g) and low dose (0.02 mg/g) TMX injected mice. High dose TMX injection caused rapid weight loss and mice reached the euthanasia criterion (20% loss of body weight) within 5 days of the injection (n = 4, solid orange). Survival of the control UbcCreERT2 mice (n = 7, dotted orange) was not affected by TMX injection. UbcCreERT2; ROSAsT mice injected with lower dose TMX exhibited significantly prolonged survival compared to high dose TMX (n = 18, solid red) and control mice were unaffected (n = 10, dotted red) (C) Multi-tissue MCV sT protein expression in UbcCreERT2; ROSAsT mice that died immediately after high dose TMX injection (< 5 days). Lower dose TMX injection induces less MCV sT protein expression in a mouse that died at day 7 after the injection as detected by immunoblotting using an antibody raised against MCV sT, CM8E6 [22]. MCV sT vector or empty vector transfected HEK293 cells were used as a positive and a negative control, respectively. Equal amounts of sT-transfected HEK293 cell lysates were loaded for normalization across different blots. Hsp70/Hsc70 was detected as a protein quality control. (D) MCV sT protein expression was maintained in tissues of UbcCreERT2; ROSAsT mice that survived over 70 days after low dose TMX injection. Immunoblots were performed as in (C). Tissue lysates from ROSAsT were used as a negative control.

Mentions: A transgenic mouse model with inducible MCV sT expression, ROSAsT, was generated in the C57BL/6 genetic background. To conditionally express MCV sT protein, codon-optimized MCV sT (sTco) cDNA was vector cloned downstream of a loxP-flanked neomycin phosphotransferase cDNA sequence with a 5’ sequence homologous to the mouse ROSA26 locus to generate ROSA26-CAG-LNL-MCVsTco (Fig 1A). ROSA26-CAG-LNL-MCVsTco was delivered by homologous recombination into the ROSA26 locus of mouse embryonic stem (ES) cells (see details in materials and methods).


Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

Shuda M, Guastafierro A, Geng X, Shuda Y, Ostrowski SM, Lukianov S, Jenkins FJ, Honda K, Maricich SM, Moore PS, Chang Y - PLoS ONE (2015)

MCV sT expression is lethal in mice.(A) Design of the ROSA26-CAG-LNL-MCVsTco, a ROSA26 knock-in construct encoding codon-optimized MCV sT with flanking loxP-STOP-Neo-loxP (LNL) sequence, recombined with the ROSA26 genomic locus. ROSA26-CAG-LNL-MCVsTco crossed with UbcCreERT2 or Atoh1CreERT2 mice allows for TMX-induced Cre recombinase excision of the loxP-STOP-Neo-loxP sequence that results in MCV sT expression under the CAG promoter. (B) MCV sT expression is lethal in UbcCreERT2; ROSAsT transgenic mice. Kaplan-Meier curve of high dose (0.2 mg/g) and low dose (0.02 mg/g) TMX injected mice. High dose TMX injection caused rapid weight loss and mice reached the euthanasia criterion (20% loss of body weight) within 5 days of the injection (n = 4, solid orange). Survival of the control UbcCreERT2 mice (n = 7, dotted orange) was not affected by TMX injection. UbcCreERT2; ROSAsT mice injected with lower dose TMX exhibited significantly prolonged survival compared to high dose TMX (n = 18, solid red) and control mice were unaffected (n = 10, dotted red) (C) Multi-tissue MCV sT protein expression in UbcCreERT2; ROSAsT mice that died immediately after high dose TMX injection (< 5 days). Lower dose TMX injection induces less MCV sT protein expression in a mouse that died at day 7 after the injection as detected by immunoblotting using an antibody raised against MCV sT, CM8E6 [22]. MCV sT vector or empty vector transfected HEK293 cells were used as a positive and a negative control, respectively. Equal amounts of sT-transfected HEK293 cell lysates were loaded for normalization across different blots. Hsp70/Hsc70 was detected as a protein quality control. (D) MCV sT protein expression was maintained in tissues of UbcCreERT2; ROSAsT mice that survived over 70 days after low dose TMX injection. Immunoblots were performed as in (C). Tissue lysates from ROSAsT were used as a negative control.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636375&req=5

pone.0142329.g001: MCV sT expression is lethal in mice.(A) Design of the ROSA26-CAG-LNL-MCVsTco, a ROSA26 knock-in construct encoding codon-optimized MCV sT with flanking loxP-STOP-Neo-loxP (LNL) sequence, recombined with the ROSA26 genomic locus. ROSA26-CAG-LNL-MCVsTco crossed with UbcCreERT2 or Atoh1CreERT2 mice allows for TMX-induced Cre recombinase excision of the loxP-STOP-Neo-loxP sequence that results in MCV sT expression under the CAG promoter. (B) MCV sT expression is lethal in UbcCreERT2; ROSAsT transgenic mice. Kaplan-Meier curve of high dose (0.2 mg/g) and low dose (0.02 mg/g) TMX injected mice. High dose TMX injection caused rapid weight loss and mice reached the euthanasia criterion (20% loss of body weight) within 5 days of the injection (n = 4, solid orange). Survival of the control UbcCreERT2 mice (n = 7, dotted orange) was not affected by TMX injection. UbcCreERT2; ROSAsT mice injected with lower dose TMX exhibited significantly prolonged survival compared to high dose TMX (n = 18, solid red) and control mice were unaffected (n = 10, dotted red) (C) Multi-tissue MCV sT protein expression in UbcCreERT2; ROSAsT mice that died immediately after high dose TMX injection (< 5 days). Lower dose TMX injection induces less MCV sT protein expression in a mouse that died at day 7 after the injection as detected by immunoblotting using an antibody raised against MCV sT, CM8E6 [22]. MCV sT vector or empty vector transfected HEK293 cells were used as a positive and a negative control, respectively. Equal amounts of sT-transfected HEK293 cell lysates were loaded for normalization across different blots. Hsp70/Hsc70 was detected as a protein quality control. (D) MCV sT protein expression was maintained in tissues of UbcCreERT2; ROSAsT mice that survived over 70 days after low dose TMX injection. Immunoblots were performed as in (C). Tissue lysates from ROSAsT were used as a negative control.
Mentions: A transgenic mouse model with inducible MCV sT expression, ROSAsT, was generated in the C57BL/6 genetic background. To conditionally express MCV sT protein, codon-optimized MCV sT (sTco) cDNA was vector cloned downstream of a loxP-flanked neomycin phosphotransferase cDNA sequence with a 5’ sequence homologous to the mouse ROSA26 locus to generate ROSA26-CAG-LNL-MCVsTco (Fig 1A). ROSA26-CAG-LNL-MCVsTco was delivered by homologous recombination into the ROSA26 locus of mouse embryonic stem (ES) cells (see details in materials and methods).

Bottom Line: To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally.Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation.Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53- setting.

View Article: PubMed Central - PubMed

Affiliation: Cancer Virology Program, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53- setting.

No MeSH data available.


Related in: MedlinePlus