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Age and insulin-like growth factor-1 impact PCNA monoubiquitination in UVB-irradiated human skin

  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Rebekah J. Hutcherson
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Ryan D. Gabbard
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
    Amber J. Castellanos
    Footnotes
    ‡ These authors contributed equally to this work.
    Affiliations
    Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA
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  • Jeffrey B. Travers
    Affiliations
    Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA

    Department of Dermatology, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA

    Dayton Veterans Affairs Medical Center, Dayton, Ohio, USA
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  • Michael G. Kemp
    Correspondence
    For correspondence: Michael G. Kemp.
    Affiliations
    Department of Pharmacology and Toxicology, Wright State University Boonshoft School of Medicine, Dayton, Ohio, USA
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  • Author Footnotes
    ‡ These authors contributed equally to this work.
Open AccessPublished:March 19, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100570
      Nonmelanoma skin cancers occur primarily in individuals over the age of 60 and are characterized by an abundance of ultraviolet (UV) signature mutations in keratinocyte DNA. Though geriatric skin removes UV photoproducts from DNA less efficiently than young adult skin, it is not known whether the utilization of other prosurvival but potentially mutagenic DNA damage tolerance systems such as translesion synthesis (TLS) is altered in older individuals. Using monoubiquitination of the replicative DNA polymerase clamp protein PCNA (proliferating cell nuclear antigen) as a biochemical marker of TLS pathway activation, we find that UVB exposure of the skin of individuals over the age of 65 results in a higher level of PCNA monoubiquitination than in the skin of young adults. Furthermore, based on previous reports showing a role for deficient insulin-like growth factor-1 (IGF-1) signaling in altered UVB DNA damage responses in geriatric human skin, we find that both pharmacological inhibition of the IGF-1 receptor (IGF-1R) and deprivation of IGF-1 potentiate UVB-induced PCNA monoubiquitination in both human skin ex vivo and keratinocytes in vitro. Interestingly, though the TLS DNA polymerase Pol eta can accurately replicate the major photoproducts induced in DNA by UV radiation, we find that it fails to accumulate on chromatin in the absence of IGF-1R signaling and that this phenotype is correlated with increased mutagenesis in keratinocytes in vitro. Thus, altered IGF-1/IGF-1R signaling in geriatric skin may predispose epidermal keratinocytes to carry out a more mutagenic form of DNA synthesis following UVB exposure.

      Keywords

      Abbreviations:

      DDT (DNA damage tolerance), HKGS (human keratinocyte growth supplement), IGF-1 (insulin-like growth factor-1), MED (minimal erythemal dose), NMSC (nonmelanoma skin cancer), PCNA (proliferating cell nuclear antigen), TLS (translesion synthesis), IGF-1R (IGF-1 receptor), IGF-1Ri (IGF-1 receptor inhibitor), PCNA-ub (monoubiquitinated PCNA)
      Nonmelanoma skin cancer (NMSC) is the most common neoplasm in humans and is primarily caused by exposures to UV wavelengths of sunlight, which induce potentially mutagenic photoproducts in DNA (
      • Brash D.E.
      UV signature mutations.
      ) if not efficiently repaired by the nucleotide excision repair system (
      • Sancar A.
      Mechanisms of DNA repair by Photolyase and excision Nuclease (Nobel Lecture).
      ). Because most skin cancers occur in individuals over the age of 60, advanced age is considered a second major risk factor for developing NMSCs (
      • Kraemer K.H.
      Sunlight and skin cancer: Another link revealed.
      ). Several reports have shown that geriatric skin exhibits a slower rate of UV photoproduct removal than the skin of young adults (
      • Xu G.
      • Snellman E.
      • Bykov V.J.
      • Jansen C.T.
      • Hemminki K.
      Effect of age on the formation and repair of UV photoproducts in human skin in situ.
      ,
      • Yamada M.
      • Udono M.U.
      • Hori M.
      • Hirose R.
      • Sato S.
      • Mori T.
      • Nikaido O.
      Aged human skin removes UVB-induced pyrimidine dimers from the epidermis more slowly than younger adult skin in vivo.
      ,
      • Freeman S.E.
      Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ.
      ,
      • Lewis D.A.
      • Travers J.B.
      • Somani A.K.
      • Spandau D.F.
      The IGF-1/IGF-1R signaling axis in the skin: A new role for the dermis in aging-associated skin cancer.
      ), which indicates that impaired excision repair may contribute to mutagenesis and ultimately carcinogenesis in older individuals.
      How the physiological microenvironment of geriatric skin impacts NMSC development in older individuals remains to be fully characterized. Fibroblasts that have undergone replication- or stress-induced senescence in vitro are known to express reduced levels of IGF-1 expression in cultured fibroblasts in vitro (
      • Lewis D.A.
      • Travers J.B.
      • Somani A.K.
      • Spandau D.F.
      The IGF-1/IGF-1R signaling axis in the skin: A new role for the dermis in aging-associated skin cancer.
      ,
      • Ferber A.
      • Chang C.
      • Sell C.
      • Ptasznik A.
      • Cristofalo V.J.
      • Hubbard K.
      • Ozer H.L.
      • Adamo M.
      • Jr C.T.R.
      • LeRoith D.
      Failure of senescent human fibroblasts to express the insulin-like growth factor-1 gene.
      ). Geriatric skin contains increased numbers of senescent dermal fibroblasts (
      • Lewis D.A.
      • Travers J.B.
      • Machado C.
      • Somani A.K.
      • Spandau D.F.
      Reversing the aging stromal phenotype prevents carcinoma initiation.
      ) and lower levels of IGF-1 expression and IGF-1 receptor (IGF-1R) activation in epidermal keratinocytes (
      • Lewis D.A.
      • Travers J.B.
      • Somani A.K.
      • Spandau D.F.
      The IGF-1/IGF-1R signaling axis in the skin: A new role for the dermis in aging-associated skin cancer.
      ). Because keratinocytes express IGF-1Rs but not IGF-1 (
      • Barreca A.
      • Luca M.
      • De, Monte P. Del
      • Bondanza S.
      • Damonte G.
      • Cariola G.
      • Marco E. Di
      • Giordano G.
      • Cancedda R.
      • Minuto F.
      In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.
      ), they are thought to be dependent on the supply from dermal fibroblasts, and several studies with keratinocytes in vitro, skin explants ex vivo, skin xenografts in vivo, and human subjects all support the idea that IGF-1 is important in how keratinocytes respond to UVB radiation (
      • Lewis D.A.
      • Travers J.B.
      • Somani A.K.
      • Spandau D.F.
      The IGF-1/IGF-1R signaling axis in the skin: A new role for the dermis in aging-associated skin cancer.
      ,
      • Kuhn C.
      • Hurwitz S.A.
      • Kumar M.G.
      • Cotton J.
      • Spandau D.F.
      Activation of the insulin-like growth factor-1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation.
      ,
      • Alkawar A.M.M.
      • Castellanos A.J.
      • Carpenter M.A.
      • Hutcherson R.J.
      • Madkhali M.A.O.
      • Johnson R.M.
      • Bottomley M.
      • Kemp M.G.
      Insulin-like growth factor-1 impacts p53 Target gene induction in UVB-irradiated keratinocytes and human skin.
      ,
      • Loesch M.M.
      • Collier A.E.
      • Southern D.H.
      • Ward R.E.
      • Tholpady S.S.
      • Lewis D.A.
      • Travers J.B.
      • Spandau D.F.
      Insulin-like growth factor-1 receptor regulates repair of ultraviolet B-induced DNA damage in human keratinocytes in vivo.
      ).
      Though geriatric skin exhibits defects in UVB photoproduct removal, there is no evidence that the skin of older individual is more prone to erythema or sunburn. Thus, geriatric skin may utilize DNA damage tolerance (DDT) pathways or other DNA damage responses to a greater extent than young adult skin. These DDT pathways encompass a variety of processes that take place at replication forks stalled at UVB photoproducts, including translesion synthesis (TLS), template switching, and repriming (
      • Pilzecker B.
      • Buoninfante O.A.
      • Jacobs H.
      DNA damage tolerance in stem cells, ageing, mutagenesis, disease and cancer therapy.
      ,
      • Sale J.E.
      Translesion DNA synthesis and mutagenesis in eukaryotes.
      ). Though significant effort has been placed on understanding these processes at the molecular level in vitro, little work has been done to determine how these pathways are utilized in human skin. This dearth of information is due in part to the lack of biochemical markers of these processes that can be exploited for use in skin tissue.
      One DDT pathway that is well recognized to impact UV-induced skin carcinogenesis is TLS, which involves the recruitment of specialized DNA polymerases to damaged DNA that can insert nucleotide opposite DNA lesions in either an error-free or an error-prone manner (
      • Sale J.E.
      Translesion DNA synthesis and mutagenesis in eukaryotes.
      ). The variant form of the skin cancer-prone disease xeroderma pigmentosum is due to mutations in the TLS DNA polymerase Pol eta (
      • Cordonnier A.M.
      • Fuchs R.P.
      Replication of damaged DNA: Molecular defect in xeroderma pigmentosum variant cells.
      ,
      • Kanao R.
      • Masutani C.
      Regulation of DNA damage tolerance in mammalian cells by post-translational modifications of PCNA.
      ,
      • Masutani C.
      • Kusumoto R.
      • Yamada A.
      • Dohmae N.
      • Yokoi M.
      • Yuasa M.
      • Araki M.
      • Iwai S.
      • Takio K.
      • Hanaoka F.
      The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.
      ), which is capable of accurately replicating UV-induced cyclobutane pyrimidine dimers (
      • Masutani C.
      • Kusumoto R.
      • Yamada A.
      • Dohmae N.
      • Yokoi M.
      • Yuasa M.
      • Araki M.
      • Iwai S.
      • Takio K.
      • Hanaoka F.
      The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.
      ,
      • Washington M.T.
      • Johnson R.E.
      • Prakash L.
      • Prakash S.
      Accuracy of lesion bypass by yeast and human DNA polymerase eta.
      ,
      • Cruet-Hennequart S.
      • Gallagher K.
      • Sokol A.M.
      • Villalan S.
      • Prendergast A.M.
      • Carty M.P.
      DNA polymerase eta, a key protein in translesion synthesis in human cells.
      ). In the absence of pol eta function, other TLS polymerases are recruited to replicate damaged DNA in a more error-prone manner. Though it is not fully clear how or why specific TLS polymerases are recruited to UV lesions in DNA, the monoubiquitination of the replicative polymerase clamp protein PCNA (proliferating cell nuclear antigen) is generally considered to be a key signal involved in the recruitment of TLS polymerases to sites of damage because these enzymes contain ubiquitin-binding domains (
      • Sale J.E.
      Translesion DNA synthesis and mutagenesis in eukaryotes.
      ,
      • Kanao R.
      • Masutani C.
      Regulation of DNA damage tolerance in mammalian cells by post-translational modifications of PCNA.
      ).
      Here we show that an antibody against the monoubiquitinated form of PCNA can be used to readily detect this important biochemical marker of TLS in UVB-irradiated human skin. We further explore the use of this new assay to explore how subject age and IGF-1 signaling impact the UVB-dependent induction of PCNA monoubiquitination in the skin. Thus, this work sheds new light on how the cellular DNA damage response may be altered in IGF-1-deficient geriatric skin to contribute to mutagenesis and NMSC development.

      Results

      PCNA monoubiquitination can be detected in UVB-irradiated human skin

      Activation of the TLS pathway is routinely assayed in cultured cells in vitro by immunoblotting cell or chromatin fractions with an anti-PCNA antibody that can detect both the unmodified protein and the monoubiquitinated form of PCNA that is shifted by ∼8-kDa shift on SDS gels (
      • Kanao R.
      • Masutani C.
      Regulation of DNA damage tolerance in mammalian cells by post-translational modifications of PCNA.
      ). To determine whether this modification can be observed in human skin tissue, we exposed human skin samples discarded during routine panniculectomies and other surgeries to increasing fluences of UVB radiation. Following a short incubation, epidermal protein lysates were prepared and analyzed by immunoblotting. As shown in Figure 1A, the ∼35-kDa unmodified form of PCNA could be readily detected with the routinely employed anti-PCNA PC10 monoclonal antibody. However, we were unable to readily or reproducibly detect the shifted, monoubiquitinated form with this antibody. In contrast, when the blot was probed with a monoclonal antibody that specifically recognizes the ubiquitinated form of PCNA, a clear band at ∼43-kDa that displayed a strong correlation with UVB dose could be observed. We note that to our knowledge, this is the first demonstration of PCNA monoubiquitination in any organ tissue.
      Figure thumbnail gr1
      Figure 1PCNA monoubiquitination can be detected in human skin epidermis. A, immunoblot analysis of epidermal protein from human skin exposed to the indicated fluences of UVB radiation. The membrane was stained with Ponceau, probed with an antibody against the monoubiquitinated form of PCNA (monoubiquitinated PCNA [PCNA-ub]; K164), and then reprobed to detect the unmodified form of PCNA. B, densitometry was used to quantify the levels of monoubiquitinated and unmodified PCNA from UVB-irradiated skin samples from three different individuals. The PCNA-ub/PCNA ratio was calculated and plotted. C, the PCNA-ub/PCNA ratios in B were normalized to the 1200 J/m2 treatment for each skin sample, which was set to an arbitrary value of 100. A variable slope, four-parameter linear regression was then used to calculate the UVB dose resulting in a half maximal response.
      Further analysis of monoubiquitinated PCNA (hereby referred to as PCNA-ub) and unmodified PCNA from several different skin donors showed there to be interindividual variability in both basal PCNA-ub and the magnitude of the response to UVB. Nonetheless, a clear UVB dose-dependent increase in PCNA-ub was observed in all three samples that were tested (Fig. 1B). By normalizing the PCNA-ub/PCNA signals to the high dose of 1200 J/m2 UVB sample and performing nonlinear regressions, UVB doses yielding a half-maximal response ranged from 500 to 700 J/m2. These UVB doses correspond to approximately twice a minimal erythemal doses (MED) for the lightly pigmented Fitzpatrick Type I-II skin used in these studies (
      • Gupta V.
      • Sharma V.K.
      Skin typing: Fitzpatrick grading and others.
      ).

      Geriatric skin displays elevated PCNA monoubiquitination following UVB exposure

      With this assay in hand, we next sought to determine how age impacts PCNA monoubiquitination in UVB-irradiated human skin. We therefore recruited young adults 21–30 years of age and geriatric adults over the age of 65 and obtained skin punch biopsies from buttock skin. One of the biopsies was exposed to 700 J/m2 UVB and incubated for 2.5 h. Epidermal protein was then analyzed by immunoblotting with PCNA-ub and PCNA antibodies, and the PCNA-ub/PCNA signal ratios were calculated. As shown in Figure 2, UV induced an increase in PCNA-ub in each of the skin samples. Interestingly, though there is significant interindividual variability (4–8-fold) in PCNA-ub levels following UVB exposure, the level of PCNA-ub was found to be increased by approximately 70% on average in geriatric subject skin in comparison to young adult skin. These results indicate that geriatric skin may utilize the TLS pathway of DNA replication to a greater degree than young adult skin, potentially to deal with the slower rate of UV photoproduct removal (
      • Xu G.
      • Snellman E.
      • Bykov V.J.
      • Jansen C.T.
      • Hemminki K.
      Effect of age on the formation and repair of UV photoproducts in human skin in situ.
      ,
      • Yamada M.
      • Udono M.U.
      • Hori M.
      • Hirose R.
      • Sato S.
      • Mori T.
      • Nikaido O.
      Aged human skin removes UVB-induced pyrimidine dimers from the epidermis more slowly than younger adult skin in vivo.
      ,
      • Freeman S.E.
      Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ.
      ,
      • Lewis D.A.
      • Travers J.B.
      • Somani A.K.
      • Spandau D.F.
      The IGF-1/IGF-1R signaling axis in the skin: A new role for the dermis in aging-associated skin cancer.
      ).
      Figure thumbnail gr2
      Figure 2Geriatric skin exhibits higher levels of PCNA monoubiquitination following UVB exposure than young adult skin. Two 5 mm punch biopsies were obtained from the buttock skin of young adults (21–30 years of age) and geriatric adults (over age 65) (n = 11 for each group), exposed to 700 J/m2 UVB radiation (or left nonirradiated), and then incubated in a 37 °C water bath for 2.5 h. Epidermal protein was analyzed for PCNA monoubiquitination as described in . The PCNA-ub/PCNA ratio was calculated and plotted for each donor. The black bar indicates the average response for each of the UVB-treated samples. An unpaired t-test was used to compare the responses in the UVB-irradiated young adult and geriatric adult skin samples.

      Inhibition of IGF-1 receptor signaling potentiates UVB-dependent induction of PCNA monoubiquitination in human skin ex vivo

      Geriatric skin exhibits numerous changes in both structure and function that may impact how epidermal cells respond to UVB radiation (
      • Branchet M.C.
      • Boisnic S.
      • Frances C.
      • Robert A.M.
      Skin thickness changes in normal aging skin.
      ,
      • Fenske N.A.
      • Lober C.W.
      Structural and functional changes of normal aging skin.
      ,
      • Cerimele D.
      • Celleno L.
      • Serri F.
      Physiological changes in ageing skin.
      ,
      • Marks R.
      Measurement of biological ageing in human epidermis.
      ). One factor that has been demonstrated to impact several UVB responses in geriatric skin is the expression of IGF-1, which, due to increased fibroblast senescence, is expressed at lower levels in geriatric skin (
      • Lewis D.A.
      • Travers J.B.
      • Somani A.K.
      • Spandau D.F.
      The IGF-1/IGF-1R signaling axis in the skin: A new role for the dermis in aging-associated skin cancer.
      ). To determine whether IGF-1 impacts PCNA monoubiquitination in human skin epidermis, human skin explants were treated topically with either vehicle (DMSO) or the IGF-1 receptor inhibitor (IGF-1Ri) AG538, which has previously been shown to serve as a convenient ex vivo model that mimics the loss of IGF-1 signaling in geriatric skin (
      • Loesch M.M.
      • Collier A.E.
      • Southern D.H.
      • Ward R.E.
      • Tholpady S.S.
      • Lewis D.A.
      • Travers J.B.
      • Spandau D.F.
      Insulin-like growth factor-1 receptor regulates repair of ultraviolet B-induced DNA damage in human keratinocytes in vivo.
      ). Skin explants from nine different middle-aged donors were then exposed to UVB radiation and analyzed for epidermal PCNA-ub levels. As shown in Figure 3, a UVB-dependent increase in PCNA-ub was observed in nearly all of the skin explant samples. Moreover, inhibiting the IGF-1R generally led to a nearly twofold higher level of PCNA-ub than in DMSO-treated skin following UVB irradiation. Thus, these results support the data in Figure 1 and indicate that the lack of IGF-1 signaling may result in a greater reliance on the TLS pathway in UVB-exposed skin.
      Figure thumbnail gr3
      Figure 3IGF-1 receptor inhibition potentiates PCNA monoubiquitination in UVB-irradiated human skin explants ex vivo. Human skin samples (n = 9) were treated topically with vehicle (DMSO) or 20 μM AG538 for 30 min before exposure to 700–800 J/m2 UVB. Skin biopsies were obtained 1–2 h later, and then PCNA monoubiquitination was measured and quantified as in and , respectively. The black bar indicates the average response for each of the UVB-treated samples, and an unpaired t-test was used to compare the responses in the UVB-irradiated DMSO- and AG538-treated skin samples.

      The loss of IGF-1 receptor signaling potentiates UVB-induced PCNA monoubiquitination in cultured keratinocytes in vitro

      To extend our findings with human skin to cultured keratinocytes in vitro, telomerase-immortalized N-TERT keratinocytes were pretreated with DMSO or the IGF-1Ri AG538 and then exposed to a low dose of UVB radiation. As shown in Figure 4A, immunoblot analysis of chromatin-enriched cell fractions demonstrated that UVB treatment induced PCNA monoubiquitination but to a greater extent in cells treated with the IGF-1Ri. Quantitation from several independent experiments confirmed these findings and showed PCNA-ub levels to be 2–3-fold higher in cells with inactive IGF-1 Rs (Fig. 4B). Similar results were observed using HaCaT keratinocytes (Fig. 4C). Lastly, inhibiting IGF-1R signaling via deprivation of IGF-1 from the cell culture medium also potentiated PCNA monoubiquitination in UVB-irradiated N-TERTs (Fig. 4D). Together, these results demonstrate that modulating IGF-1/IGF-1R signaling in keratinocytes in vitro results in a similar response to UVB radiation as total skin epidermal cells ex vivo.
      Figure thumbnail gr4
      Figure 4Loss of IGF-1 receptor signaling potentiates PCNA monoubiquitination in UVB-keratinocytes in vitro. A, Telomerase-immortalized N-TERT keratinocytes were treated with vehicle (DMSO) or 10 μM AG538 for 30 min before exposure to 50 J/m2 UVB radiation. Cells were harvested 30 min later and fractionated to enrich for chromatin-associated proteins, which were analyzed by immunoblotting. Short and long exposures of the blot are shown. B, the ratio of PCNA-ub to PCNA from five independent experiments was calculated for each experiment and then blotted with the average response. Error bars indicate the SEM. C, HaCaT keratinocytes were treated as described in A. Quantitation of relative the PCNA-ub/PCNA ration is shown below the blot. D, N-TERTs were cultured in medium lacking or containing IGF-1 for 24 h prior to UVB exposure and processing as in A. The fold change in PCNA-ub/PCNA was calculated from three independent experiments.

      Loss of IGF-1 signaling is associated with mutagenesis and a failure of pol eta to accumulate on UVB-damaged chromatin

      The monoubiquitination of PCNA serves as a signal for the recruitment of specialized TLS polymerases to damaged DNA (
      • Sale J.E.
      Translesion DNA synthesis and mutagenesis in eukaryotes.
      ,
      • Kanao R.
      • Masutani C.
      Regulation of DNA damage tolerance in mammalian cells by post-translational modifications of PCNA.
      ). In the case of UVB-induced DNA damage, the TLS polymerase Pol eta is capable of accurately replicating the common thymine dimers induced by UV radiation. In response to UVC radiation, Pol eta has been found to accumulate on chromatin in cultured cells in vitro, which can be assayed by immunoblotting chromatin-enriched cell fractions (
      • Lerner L.K.
      • Francisco G.
      • Soltys D.T.
      • Rocha C.R.
      • Quinet A.
      • Vessoni A.T.
      • Castro L.P.
      • David T.I.
      • Bustos S.O.
      • Strauss B.E.
      • Gottifredi V.
      • Stary A.
      • Sarasin A.
      • Chammas R.
      • Menck C.F.
      Predominant role of DNA polymerase eta and p53-dependent translesion synthesis in the survival of ultraviolet-irradiated human cells.
      ). As shown in Figure 5, A and B, we observed that Pol eta was enriched by 2–3-fold on chromatin within 30 min of exposure of N-TERTs to a low dose of UVB radiation. However, this enrichment was nearly completely abrogated by treatment with the IGF-1Ri AG538. Importantly, IGF-1R inhibition did not affect the amount of Pol eta present in the soluble fraction of the cells (Fig. 5A).
      Figure thumbnail gr5
      Figure 5Loss of IGF-1 signaling is associated with a defect in Pol eta accumulation on UVB-damaged chromatin. A, Chromatin lysates from N-TERTs treated with DMSO or AG538 and exposed to UVB as in A were analyzed by immunoblotting. B and C, the fold change in TLS polymerase level on chromatin was calculated and graphed from three independent experiments. D, Soluble lysates from N-TERTs were examined by immunoblotting. E, N-TERTs grown in the absence or presence of IGF-1 for 24 h were exposed to 50 J/m2 UVB, incubated for 30 min, and then chromatin fractions were analyzed for Pol eta protein levels. F, the fold change in chromatin-associated Pol eta levels was calculated in three independent experiments performed as in C with N-TERTs and HaCaT cells.
      In the absence of Pol eta function, other TLS polymerases are likely required to replicate unrepaired UVB photoproducts. Consistent with this hypothesis, we observed that both Pol iota and Rev1, which play recognized roles in UV mutagenesis (
      • Sale J.E.
      Translesion DNA synthesis and mutagenesis in eukaryotes.
      ), become modestly enriched on chromatin in UVB-irradiated keratinocytes in which IGF-1 signaling has been inhibited (Fig. 5, A and C). A similar defect in Pol eta recruitment to chromatin was observed when N-TERTs and HaCaT keratinocytes were deprived of IGF-1 in the culture medium (Fig. 5, D, F and E). Thus, the major TLS polymerase thought to be responsible for accurately replicating unrepaired UV photoproducts fails to associate with UV-damaged chromatin following UVB exposure in cultured keratinocytes in vitro when IGF-1 signaling is disrupted.
      Increased PCNA monoubiquitination coupled with the failure to recruit Pol eta to damaged chromatin suggests that other, potentially more mutagenic DNA polymerases may be involved in replicating unrepaired UV lesions when IGF-1 signaling is lost. To determine whether there is a higher level of mutagenesis under these conditions, we treated UVB-irradiated HaCaT cells with 6-thioguanine to select for cells with mutations at the HPRT locus, which imparts resistance to the drug (
      • Johnson G.E.
      Mammalian cell HPRT gene mutation assay: Test methods.
      ). As shown in Figure 6, HaCaT cells treated with DMSO vehicle exhibited a low mutation frequency of 10 per million cells. In contrast, when cells were treated with the IGF-1Ri AG538, the mutation rate was increased approximately threefold. We conclude from these experiments that the loss of IGF-1 signaling increases mutagenesis induced by UVB radiation.
      Figure thumbnail gr6
      Figure 6Loss of IGF-1 signaling is associated increased mutagenesis in UVB-irradiated keratinocytes. HaCaT cells were treated with DMSO or the IGF-1R inhibitor AG538 as in , and media was replaced with drug-free medium 5 h after UVB-exposure. Five days later, cells were replated and selected with 6-thioguanine to detect cells with mutations at the HPRT locus. The graphs show the average (±SEM) number of mutant cells (per million cells plated) along with the individual experimental values, and an unpaired t-test was used to analyze the results.

      Discussion

      Though the structure and physiological microenvironment of the skin are known to change over the course of the lifetime (
      • Branchet M.C.
      • Boisnic S.
      • Frances C.
      • Robert A.M.
      Skin thickness changes in normal aging skin.
      ,
      • Fenske N.A.
      • Lober C.W.
      Structural and functional changes of normal aging skin.
      ,
      • Cerimele D.
      • Celleno L.
      • Serri F.
      Physiological changes in ageing skin.
      ,
      • Marks R.
      Measurement of biological ageing in human epidermis.
      ) and to lead to an increased risk of NMSC development in older individuals (
      • Kraemer K.H.
      Sunlight and skin cancer: Another link revealed.
      ), the mechanisms responsible for this effect remain to be fully elucidated. Historically, most experimental studies of UVB-irradiated human skin have been restricted to measurements of UVB photoproduct levels at various time points following UVB exposure. Though this information is valuable, additional work is necessary to understand whether the plethora of DNA damage responses that govern the cell fate following UVB exposure are impacted by age and other factors of the skin.
      Here we showed for the first time that PCNA monoubiquitination, a posttranslational modification associated with the activation of the TLS pathway and the recruitment of specialized DNA polymerases to sites of DNA damage (
      • Sale J.E.
      Translesion DNA synthesis and mutagenesis in eukaryotes.
      ,
      • Kanao R.
      • Masutani C.
      Regulation of DNA damage tolerance in mammalian cells by post-translational modifications of PCNA.
      ), can be readily detected in human skin epidermis following UVB exposure (Fig. 1). Moreover, the observation that this response is potentiated by advanced age (Fig. 2) and the loss of IGF-1R signaling (Fig. 3) suggest that the age-dependent decline in IGF-1 expression in the skin (
      • Lewis D.A.
      • Travers J.B.
      • Somani A.K.
      • Spandau D.F.
      The IGF-1/IGF-1R signaling axis in the skin: A new role for the dermis in aging-associated skin cancer.
      ,
      • Alkawar A.M.M.
      • Castellanos A.J.
      • Carpenter M.A.
      • Hutcherson R.J.
      • Madkhali M.A.O.
      • Johnson R.M.
      • Bottomley M.
      • Kemp M.G.
      Insulin-like growth factor-1 impacts p53 Target gene induction in UVB-irradiated keratinocytes and human skin.
      ) impacts several canonical DNA damage responses in addition to the rate of removal of UVB photoproducts (
      • Loesch M.M.
      • Collier A.E.
      • Southern D.H.
      • Ward R.E.
      • Tholpady S.S.
      • Lewis D.A.
      • Travers J.B.
      • Spandau D.F.
      Insulin-like growth factor-1 receptor regulates repair of ultraviolet B-induced DNA damage in human keratinocytes in vivo.
      ) and the activation of ATR kinase signaling (
      • Kemp M.G.
      • Spandau D.F.
      • Simman R.
      • Travers J.B.
      Insulin-like growth factor 1 receptor signaling is required for Optimal ATR-CHK1 kinase signaling in ultraviolet B (UVB)-irradiated human keratinocytes.
      ).
      Moreover, our observation that the TLS polymerase Pol eta is not efficiently recruited to UVB-damaged chromatin in IGF-1-deficient keratinocytes in vitro (Fig. 5) suggests that other, potentially more mutagenic DNA polymerases are likely responsible for replicating UVB-damaged DNA under these conditions. Pol eta recruitment to sites of DNA damage has been reported to require both ATR kinase and Protein Kinase C signaling (
      • Gohler T.
      • Sabbioneda S.
      • Green C.M.
      • Lehmann A.R.
      ATR-mediated phosphorylation of DNA polymerase eta is needed for efficient recovery from UV damage.
      ,
      • Andrade-Lima L.C.
      • Andrade L.N.
      • Menck C.F.
      ATR suppresses apoptosis after UVB irradiation by controlling both translesion synthesis and alternative tolerance pathways.
      ,
      • Chen Y.W.
      • Cleaver J.E.
      • Hatahet Z.
      • Honkanen R.E.
      • Chang J.Y.
      • Yen Y.
      • Chou K.M.
      Human DNA polymerase eta activity and translocation is regulated by phosphorylation.
      ), and thus it is possible that the defects in ATR signaling that we (
      • Kemp M.G.
      • Spandau D.F.
      • Simman R.
      • Travers J.B.
      Insulin-like growth factor 1 receptor signaling is required for Optimal ATR-CHK1 kinase signaling in ultraviolet B (UVB)-irradiated human keratinocytes.
      ) and others (
      • Fernandez T.L.
      • Lonkhuyzen D. R. Van
      • Dawson R.A.
      • Kimlin M.G.
      • Upton Z.
      Insulin-like growth factor-I and UVB photoprotection in human keratinocytes.
      ) have reported in IGF-1-deficient keratinocytes in vitro contribute to the aberrant recruitment. Precisely how IGF-1/IGF-1R signaling impacts PCNA ubiquitination and TLS polymerase recruitment remains to be better defined. In addition to the regulation of AKT and MAPK signaling (
      • Siddle K.
      Signalling by insulin and IGF receptors: Supporting acts and new players.
      ), which could directly or indirectly impact various aspects of cell metabolism, recent studies indicate that the IGF-1R can directly interact with and phosphorylate PCNA in some cancer cell lines (
      • Waraky A.
      • Lin Y.
      • Warsito D.
      • Haglund F.
      • Aleem E.
      • Larsson O.
      Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.
      ,
      • Yang C.
      • Zhang Y.
      • Chen Y.
      • Ragaller F.
      • Liu M.
      • Corvigno S.
      • Dahlstrand H.
      • Carlson J.
      • Chen Z.
      • Näsman A.
      • Waraky A.
      • Lin Y.
      • Larsson O.
      • Haglund F.
      Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers.
      ). Thus, additional work is necessary to understand how the IGF-1R impacts genome stability in UVB-irradiated keratinocytes in vitro and within the context of human skin.
      We would also note that we recently showed that Pol eta fails to be properly induced at the transcriptional level following UVB exposure in human keratinocytes in vitro and skin ex vivo when IGF-1 signaling is abrogated (
      • Alkawar A.M.M.
      • Castellanos A.J.
      • Carpenter M.A.
      • Hutcherson R.J.
      • Madkhali M.A.O.
      • Johnson R.M.
      • Bottomley M.
      • Kemp M.G.
      Insulin-like growth factor-1 impacts p53 Target gene induction in UVB-irradiated keratinocytes and human skin.
      ). Together, these results suggest that chronically sun-exposed regions of geriatric skin may be particularly vulnerable to mutagenic forms of DNA synthesis due to an increased reliance on TLS via elevated PCNA monoubiquitination and an inability to recruit Pol eta to sites of DNA damage. However, future studies will be needed to test this hypothesis. Lastly, because dermal wounding modes of skin rejuvenation have been shown to restore IGF-1 expression in geriatric skin (
      • Lewis D.A.
      • Travers J.B.
      • Machado C.
      • Somani A.K.
      • Spandau D.F.
      Reversing the aging stromal phenotype prevents carcinoma initiation.
      ,
      • Spandau D.F.
      • Lewis D.A.
      • Somani A.K.
      • Travers J.B.
      Fractionated laser resurfacing corrects the inappropriate UVB response in geriatric skin.
      ,
      • Travers J.B.
      • Spandau D.F.
      • Lewis D.A.
      • Machado C.
      • Kingsley M.
      • Mousdicas N.
      • Somani A.K.
      Fibroblast senescence and squamous cell carcinoma: How wounding therapies could be protective.
      ), it will be important to determine whether these clinical interventions improve the ability of geriatric skin to accurately replicate unrepaired UVB photoproducts.

      Experimental procedures

      Human skin samples

      Experiments with human skin involved the skin obtained from two different sources. In the first study, two 5 mm skin punch biopsies were obtained from each of 11 young adults (21–30 years of age) and 11 geriatric adults (over 65 years of age) with Fitzpatrick Type I-II skin. This human subject research protocol was approved by the Wright State University Institutional Review Board and abides by the Declaration of Helsinki principles. Within 30 min of excision, one biopsy was then exposed to 700 J/m2 UVB with a Philips F20T12 UVB bulb at a dose rate of 5 J/m2/sec. The other biopsy was sham-treated, and then both biopsies were incubated for 2.5 h incubation in a 37 °C water bath before freezing in liquid nitrogen. Epidermal lysates were prepared in RIPA buffer containing protease and phosphatase inhibitors and then 3 μg of each sample was separated by SDS-PAGE and analyzed by immunoblotting. In the second set of experiments, discarded, deidentified skin from panniculectomies and other surgical procedures was treated topically with DMSO vehicle or 20 μM AG538 for 30 min before exposure to UVB radiation and incubation for 1–2 h in a water bath. Skin punch biopsies (5–6 mm) were then obtained and snap frozen in liquid nitrogen.

      Cell culture

      N-TERT cells were cultured as previously described in EpiLife medium containing human keratinocyte growth supplement (HKGS) and penicillin/streptomycin (
      • Kemp M.G.
      • Spandau D.F.
      • Simman R.
      • Travers J.B.
      Insulin-like growth factor 1 receptor signaling is required for Optimal ATR-CHK1 kinase signaling in ultraviolet B (UVB)-irradiated human keratinocytes.
      ). HaCaT cells were usually grown in DMEM containing FBS and penicillin/streptomycin, but HaCaT cells were also adapted for growth in EpiLife medium in certain experiments in which cells were cultured in EpiLife medium with HKGS lacking or containing IGF-1. DMSO or the IGF-1R inhibitor AG538 was added to medium as previously described (
      • Kemp M.G.
      • Spandau D.F.
      • Simman R.
      • Travers J.B.
      Insulin-like growth factor 1 receptor signaling is required for Optimal ATR-CHK1 kinase signaling in ultraviolet B (UVB)-irradiated human keratinocytes.
      ). Cells were irradiated with the same UVB light source as described for human skin.

      Immunoblotting

      To prepare skin epidermal samples for immunoblot analysis, the punch biopsies were briefly heated in a water bath at 60–70 °C for 6 s and then placed in an ice bath for 9 s. A curette was then used to separate the dermis from the epidermis, which was then sonicated in RIPA buffer and centrifuged for 20 min at maximum speed in a microcentrifuge at 4 °C. To prepare protein lysates from cultured keratinocytes, cells were extracted twice with a modified cytoskeletal buffer (10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, and 0.1% Triton X-100). The soluble or chromatin-enriched fractions were then used for immunoblotting as described in the figure legend. Cell and epidermal lysates were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and then stained with Ponceau S to ensure equal protein loading. Following washing with TBST (Tris-buffered saline containing 0.1% Tween-20) and blocking in 5% milk in TBST, blots were probed with 1:2000 or 1:5000 dilutions of antibodies against ubiquityl-PCNA (Lys 164; Cell Signaling #13439), PCNA (Santa Cruz sc-56), Pol eta (Santa Cruz sc-17770), Pol iota (GeneTex GTX112137), and Rev1 (Santa Cruz sc-393022). After washing, the blots were probed with HRP-coupled anti-mouse or anti-rabbit IgG (ThermoFisher) secondary antibodies for 1–2 h at room temperature. Chemiluminescence was visualized with either Clarity Western ECL substrate (Bio-Rad) or SuperSignal West Femto substrate (Thermo Scientific) using a Molecular Imager Chemi-Doc XRS+ imaging system (Bio-Rad). Signals in the linear range of detection were quantified by densitometry using Image Lab (Bio-Rad) and normalized as previously described (
      • Kemp M.G.
      • Krishnamurthy S.
      • Kent M.N.
      • Schumacher D.L.
      • Sharma P.
      • Excoffon K.J.D.A.
      • Travers J.B.
      Spironolactone Depletes the XPB protein and Inhibits DNA damage responses in UVB-irradiated human skin.
      ) or calculated as fold changes in UVB-irradiated samples relative to the nonirradiated sample in each experiment. Unpaired t-tests were used to compare the UVB-dependent changes in protein levels between the treatment groups.

      HPRT mutagenesis assays

      HaCaT cells were pretreated with DMSO or the IGF-1R inhibitor AG538 for 30 min before exposure to 50 J/m2 UVB. Media was replaced with drug-free medium 5 h later. Following a 5 days of cell proliferation, cells were replated for analysis of mutagenesis at the HPRT locus by selection with 4 μg/ml 6-thioguanine for 2 weeks (
      • Johnson G.E.
      Mammalian cell HPRT gene mutation assay: Test methods.
      ). Surviving colonies were stained with crystal violet to calculate the mutant frequency. An ANOVA was run to analyze the data sets.

      Data availability

      All data are provided in the article and/or are available upon request to [email protected]

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank the WSU Proteome Analysis Laboratory and Center for Genomics Research for the use of equipment to carry out this work.

      Author contributions

      M. G. K. designed and supervised the project. R. D. G and J. B. T. collected skin punch biopsies from human subjects. R. J. H., A. J. C., and M. G. K. performed the experiments. M. G. K. analyzed the data and wrote the initial draft of the article. All the authors provided comments on the article.

      Funding and additional information

      This work was supported by start-up funding by Wright State University (to M. G. K.) and by grants from the National Institute of General Medical Sciences (GM130583 to M. G. K.), the National Institute on Aging (AG048946 to J. B. T.), and the Veterans Administration (Clinical VA Merit Award 1101CX000809 to J. B. T.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the US Veterans Administration.

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