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Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture.

Hoskins EE, Morris TA, Higginbotham JM, Spardy N, Cha E, Kelly P, Williams DA, Wikenheiser-Brokamp KA, Duensing S, Wells SI - Oncogene (2008)

Bottom Line: Organotypic raft cultures exhibited DNA repair-associated defects with more 53BP1 foci and TdT-mediated dNTP nick end labeling-positive cells over their corrected counterparts.Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers.Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.

ABSTRACT
Fanconi anemia (FA) is a recessive genome instability syndrome characterized by heightened cellular sensitivity to DNA damage, aplastic anemia and cancer susceptibility. Leukemias and squamous cell carcinomas (SCCs) are the most predominant FA-associated cancers, with the latter exhibiting markedly early disease onset and aggressiveness. Although studies of hematopoietic cells derived from FA patients have provided much insight into bone marrow deficiencies and leukemogenesis, molecular transforming events in FA-deficient keratinocytes, which are the cell type of origin for SCC, are poorly understood. We describe here the growth and molecular properties of FANCA-deficient versus FANCA-corrected HPV E6/E7 immortalized keratinocytes in monolayer and organotypic epithelial raft culture. In response to DNA damage, FANCA-deficient patient-derived keratinocyte cultures displayed a G2/M phase arrest, senescence and apoptosis. Organotypic raft cultures exhibited DNA repair-associated defects with more 53BP1 foci and TdT-mediated dNTP nick end labeling-positive cells over their corrected counterparts. Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers. The observed FANCA-mediated suppression of hyperplasia correlated with the detection of fewer cells transiting through the cell cycle in the absence of gross differentiation abnormalities or apoptotic differences. Importantly, the knockdown of either FANCA or FANCD2 in HPV-positive keratinocytes was sufficient for increasing epithelial hyperplasia. Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.

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Suppression of HPV associated epithelial hyperplasia by FANCA complementationA. Organotypic raft sections were stained with hematoxylin and eosin for morphological evaluation (top panel). Hyperplasia as observed in the FANCA deficient rafts was additionally quantitated by nuclear counts. The data are represented in the graph on the right. Immunofluorescence detection of E-cadherin, K14 and K10 are shown in the second, third and fourth panel from the top, respectively. Two asterisks indicate a p value of less than 0.01. B. Keratinocytes were immortalized by transfection with HPV16 genomic DNA as described in (Hoskins et al., 2008). Subsequent complementation, sorting and organotypic raft generation for morphological evaluation was carried out as described for A. The data are quantitated by nuclear counts, Fig. 5B depicts quantitation of Ki67 and BrdU positivity. C. HPV-NIKs were infected with nontargeting control or FANCA knockdown vectors, selected in puromycin and used for organotypic raft generation. Western blot analysis demonstrates cellular inability to respond to DNA damage by the monoubiquitination of FANCD2 (compare lanes 4 and 2). Morphological evaluation was carried out following H&E staining as in B.
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Figure 4: Suppression of HPV associated epithelial hyperplasia by FANCA complementationA. Organotypic raft sections were stained with hematoxylin and eosin for morphological evaluation (top panel). Hyperplasia as observed in the FANCA deficient rafts was additionally quantitated by nuclear counts. The data are represented in the graph on the right. Immunofluorescence detection of E-cadherin, K14 and K10 are shown in the second, third and fourth panel from the top, respectively. Two asterisks indicate a p value of less than 0.01. B. Keratinocytes were immortalized by transfection with HPV16 genomic DNA as described in (Hoskins et al., 2008). Subsequent complementation, sorting and organotypic raft generation for morphological evaluation was carried out as described for A. The data are quantitated by nuclear counts, Fig. 5B depicts quantitation of Ki67 and BrdU positivity. C. HPV-NIKs were infected with nontargeting control or FANCA knockdown vectors, selected in puromycin and used for organotypic raft generation. Western blot analysis demonstrates cellular inability to respond to DNA damage by the monoubiquitination of FANCD2 (compare lanes 4 and 2). Morphological evaluation was carried out following H&E staining as in B.

Mentions: In addition to evidence of DNA damage suppression in Fig. 3, the FANCA complemented raft exhibited decreased hyperplasia upon morphological comparison (Fig. 4). Hematoxylin and eosin staining demonstrated increased raft thickness and the presence of additional spinous cell layers in the FANCA deficient compared to the complemented rafts. Similar phenotypes were noted in two additional, independently derived epithelial rafts obtained from the same cell populations (data not shown). Hyperplasia was reflected by increased numbers of nuclei in the suprabasal compartment. Closer morphological examination revealed approximately equal numbers of dysplastic and dyskeratotic cells, likely related to E6/E7 expression, in both rafts (data not shown). Epithelial differentiation and maturation properties were also similar as verified by comparable patterns of expression of the basal cell marker K14 and the suprabasal cell marker K10 by immunofluorescence (Fig. 4A, bottom two panels). Detection of the adherens junction component E-cadherin revealed more E-cadherin positive cell layers, likely a reflection of increased raft thickness, in the FANCA deficient epithelium. However, we did not note obvious E-cadherin mis-localization and/or expression at a single cell level, indicating similar cell-cell contact properties in the FANCA deficient versus complemented epithelium. A marked reduction in hyperplasia was also observed upon FANCA complementation of HPV16 episome-immortalized FA patient keratinocytes (Fig. 4B). Conversely, we detected an increase in hyperplasia following lentiviral knockdown of FANCA in HPV positive normal human keratinocytes (NIKs) and subsequent raft generation (Fig. 4C), thus ruling out the possibility that clonal selection might have been associated with the observed differences between FANCA deficient versus sufficient patient-derived keratinocytes. We cannot fully exclude the possibility that increased hyperplasia is a consequence of subtle cellular adaptation to FA loss that was undetectable in 2D culture. However, if proliferative gains and epithelial hyperplasia are adaptive in nature, our data would support that they remain reversible through the re-instatement of functional FA pathways by complementation.


Fanconi anemia deficiency stimulates HPV-associated hyperplastic growth in organotypic epithelial raft culture.

Hoskins EE, Morris TA, Higginbotham JM, Spardy N, Cha E, Kelly P, Williams DA, Wikenheiser-Brokamp KA, Duensing S, Wells SI - Oncogene (2008)

Suppression of HPV associated epithelial hyperplasia by FANCA complementationA. Organotypic raft sections were stained with hematoxylin and eosin for morphological evaluation (top panel). Hyperplasia as observed in the FANCA deficient rafts was additionally quantitated by nuclear counts. The data are represented in the graph on the right. Immunofluorescence detection of E-cadherin, K14 and K10 are shown in the second, third and fourth panel from the top, respectively. Two asterisks indicate a p value of less than 0.01. B. Keratinocytes were immortalized by transfection with HPV16 genomic DNA as described in (Hoskins et al., 2008). Subsequent complementation, sorting and organotypic raft generation for morphological evaluation was carried out as described for A. The data are quantitated by nuclear counts, Fig. 5B depicts quantitation of Ki67 and BrdU positivity. C. HPV-NIKs were infected with nontargeting control or FANCA knockdown vectors, selected in puromycin and used for organotypic raft generation. Western blot analysis demonstrates cellular inability to respond to DNA damage by the monoubiquitination of FANCD2 (compare lanes 4 and 2). Morphological evaluation was carried out following H&E staining as in B.
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Figure 4: Suppression of HPV associated epithelial hyperplasia by FANCA complementationA. Organotypic raft sections were stained with hematoxylin and eosin for morphological evaluation (top panel). Hyperplasia as observed in the FANCA deficient rafts was additionally quantitated by nuclear counts. The data are represented in the graph on the right. Immunofluorescence detection of E-cadherin, K14 and K10 are shown in the second, third and fourth panel from the top, respectively. Two asterisks indicate a p value of less than 0.01. B. Keratinocytes were immortalized by transfection with HPV16 genomic DNA as described in (Hoskins et al., 2008). Subsequent complementation, sorting and organotypic raft generation for morphological evaluation was carried out as described for A. The data are quantitated by nuclear counts, Fig. 5B depicts quantitation of Ki67 and BrdU positivity. C. HPV-NIKs were infected with nontargeting control or FANCA knockdown vectors, selected in puromycin and used for organotypic raft generation. Western blot analysis demonstrates cellular inability to respond to DNA damage by the monoubiquitination of FANCD2 (compare lanes 4 and 2). Morphological evaluation was carried out following H&E staining as in B.
Mentions: In addition to evidence of DNA damage suppression in Fig. 3, the FANCA complemented raft exhibited decreased hyperplasia upon morphological comparison (Fig. 4). Hematoxylin and eosin staining demonstrated increased raft thickness and the presence of additional spinous cell layers in the FANCA deficient compared to the complemented rafts. Similar phenotypes were noted in two additional, independently derived epithelial rafts obtained from the same cell populations (data not shown). Hyperplasia was reflected by increased numbers of nuclei in the suprabasal compartment. Closer morphological examination revealed approximately equal numbers of dysplastic and dyskeratotic cells, likely related to E6/E7 expression, in both rafts (data not shown). Epithelial differentiation and maturation properties were also similar as verified by comparable patterns of expression of the basal cell marker K14 and the suprabasal cell marker K10 by immunofluorescence (Fig. 4A, bottom two panels). Detection of the adherens junction component E-cadherin revealed more E-cadherin positive cell layers, likely a reflection of increased raft thickness, in the FANCA deficient epithelium. However, we did not note obvious E-cadherin mis-localization and/or expression at a single cell level, indicating similar cell-cell contact properties in the FANCA deficient versus complemented epithelium. A marked reduction in hyperplasia was also observed upon FANCA complementation of HPV16 episome-immortalized FA patient keratinocytes (Fig. 4B). Conversely, we detected an increase in hyperplasia following lentiviral knockdown of FANCA in HPV positive normal human keratinocytes (NIKs) and subsequent raft generation (Fig. 4C), thus ruling out the possibility that clonal selection might have been associated with the observed differences between FANCA deficient versus sufficient patient-derived keratinocytes. We cannot fully exclude the possibility that increased hyperplasia is a consequence of subtle cellular adaptation to FA loss that was undetectable in 2D culture. However, if proliferative gains and epithelial hyperplasia are adaptive in nature, our data would support that they remain reversible through the re-instatement of functional FA pathways by complementation.

Bottom Line: Organotypic raft cultures exhibited DNA repair-associated defects with more 53BP1 foci and TdT-mediated dNTP nick end labeling-positive cells over their corrected counterparts.Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers.Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.

ABSTRACT
Fanconi anemia (FA) is a recessive genome instability syndrome characterized by heightened cellular sensitivity to DNA damage, aplastic anemia and cancer susceptibility. Leukemias and squamous cell carcinomas (SCCs) are the most predominant FA-associated cancers, with the latter exhibiting markedly early disease onset and aggressiveness. Although studies of hematopoietic cells derived from FA patients have provided much insight into bone marrow deficiencies and leukemogenesis, molecular transforming events in FA-deficient keratinocytes, which are the cell type of origin for SCC, are poorly understood. We describe here the growth and molecular properties of FANCA-deficient versus FANCA-corrected HPV E6/E7 immortalized keratinocytes in monolayer and organotypic epithelial raft culture. In response to DNA damage, FANCA-deficient patient-derived keratinocyte cultures displayed a G2/M phase arrest, senescence and apoptosis. Organotypic raft cultures exhibited DNA repair-associated defects with more 53BP1 foci and TdT-mediated dNTP nick end labeling-positive cells over their corrected counterparts. Interestingly, together with reduced rates of DNA damage, FA correction resulted in a marked decrease in epithelial thickness and the presence of fewer cell layers. The observed FANCA-mediated suppression of hyperplasia correlated with the detection of fewer cells transiting through the cell cycle in the absence of gross differentiation abnormalities or apoptotic differences. Importantly, the knockdown of either FANCA or FANCD2 in HPV-positive keratinocytes was sufficient for increasing epithelial hyperplasia. Our findings support a new role for FA pathways in the maintenance of differentiation-dependent cell cycle exit, with the implication that FA deficiencies may contribute to the high risk of FA patients for developing HPV-associated SCC.

Show MeSH
Related in: MedlinePlus