Davina A. Lewis

Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects.

Abstract Lewis, D. A., Mayhugh, B. M., Qin, Y., Trott, K. and Mendonca, M. S. Production of Delayed Death and Neoplastic Transformation in CGL1 Cells by Radiation-Induced Bystander Effects. Radiat. Res. 156, 251–258 (2001). Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence α particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 ± 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 × 10–6 in unirradiated controls to 2.3 × 10–5 (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.

The IGF-1/IGF-1R signaling axis in the skin: a new role for the dermis in aging-associated skin cancer.

The appropriate response of human keratinocytes to ultraviolet-B (UVB) is dependent on the activation status of the insulin-like growth factor 1 (IGF-1) receptor. Keratinocytes grown in conditions in which the IGF-1 receptor is inactive inappropriately replicate in the presence of UVB-induced DNA damage. In human skin, epidermal keratinocytes do not express IGF-1, and hence the IGF-1 receptor on keratinocytes is activated by IGF-1 secreted from dermal fibroblasts. We now show that the IGF-1 produced by human fibroblasts is essential for the appropriate UVB response of keratinocytes. Furthermore, the expression of IGF-1 is silenced in senescent fibroblasts in vitro. Using quantitative reverse transcriptase–PCR and immunohistochemisty, we can show that IGF-1 expression is also silenced in geriatric dermis in vivo. The diminished IGF-1 expression in geriatric skin correlates with an inappropriate UVB response in geriatric volunteers. Finally, the appropriate UVB response is restored in geriatric skin in vivo through pretreatment with exogenous IGF-1. These studies provide further evidence for a role of the IGF-1 receptor (IGF-1R) in suppressing UVB-induced carcinogenesis, suggest that fibroblasts have a critical role in maintaining appropriate activation of the keratinocyte IGF-1R, and imply that reduced expression of IGF-1 in geriatric skin could be an important component in the development of aging-related non-melanoma skin cancer.

UVB-induced Senescence in Human Keratinocytes Requires a Functional Insulin-like Growth Factor-1 Receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21(CDKN1A). Furthermore, IGF-1R-dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R-dependent premature senescence.

Decreased incidence of nonmelanoma skin cancer in patients with type 2 diabetes mellitus using insulin: a pilot study.

Background  In order to prevent the propagation of genetic mutations, human keratinocytes irradiated with ultraviolet (UV) B light in vitro undergo premature stress‐induced senescence or apoptosis. This response to UVB irradiation is dependent on the functional activation of the insulin‐like growth factor‐1 receptor (IGF‐1R). Based on this in vitro functional data, we hypothesized that the increased serum levels of insulin in patients with type 2 diabetes may activate the IGF‐1R in skin and lead to a decreased frequency of skin cancer in these patients.

Augmentation of Chemotherapy-Induced Cytokine Production by Expression of the Platelet-Activating Factor Receptor in a Human Epithelial Carcinoma Cell Line

In addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in tumor cells. Moreover, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors (PAF-R) linked to cytokine production, it is not known whether PAF is involved in chemotherapeutic agent-induced cytokine production. These studies examined the role of the PAF system in chemotherapy-mediated cytokine production using a model system created by retroviral-mediated transduction of the PAF-R-negative human epidermal carcinoma cell line KB with the human PAF-R. The presence of the PAF-R in KB cells resulted in augmentation of the production of cytokines IL-8 and TNF-α induced by the chemotherapeutic agents etoposide and mitomycin C. These effects were specific for the PAF-R, as expression of the G protein-coupled receptor for fMLP did not affect chemotherapeutic agent-induced cytokine production. Moreover, ablation of the native PAF-R in the epithelial cell line HaCaT using an inducible antisense PAF-R strategy inhibited etoposide-induced cytokine production. Oxidative stress and the transcription factor NF-κB were found to be involved in this augmentative effect, because it was mimicked by the oxidant tert-butyl-hydroperoxide, which was blocked both by antioxidants and by inhibition of the NFκB pathway using a super-repressor IκBM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-κB-dependent process.

UVB activation of NF-κB in normal human keratinocytes occurs via a unique mechanism

The transcription factor nuclear factor-κB (NF-κB) is comprised of a family of proteins that are implicated in a wide variety of cellular functions, including the control of cell proliferation, cell survival, and cellular differentiation. Although NF-κB is activated in response to inflammatory signals or cellular stress, in the skin NF-κB is also implicated to play a role in normal epidermal homeostasis. Often the cellular consequences of NF-κB activation are dependent on the specific triggering stimuli. Thus, we have compared the activation mechanism and the function of NF-κB following two common stimuli of normal human keratinocytes, inflammatory mediators (tumor necrosis factor alpha (TNFα)), and cellular stress (ultraviolet light B (UVB) irradiation). These experiments indicate that although both TNFα and UVB stimulate NF-κB DNA-binding activity in normal human keratinocytes, the mechanisms of NF-κB activation by each stimulus is different. In contrast to the NF-κB response following TNFα, activation of NF-κB by UVB is independent of IκBα degradation. Analyses of NF-κB-dependent gene expression following TNFα or UVB treatment demonstrate that each of these stimulatory signals results in a specific subset of genes that are activated or repressed. These studies provide further evidence of the stimuli and cell-type specific nature of NF-κB function.

UVB-induced apoptosis in normal human keratinocytes: role of the erbB receptor family

Exposure of human keratinocytes to ultraviolet B (UVB) light leads to the activation of a variety of cell-surface receptors; however, the biologic consequences of these activated receptors are still unclear. It was previously reported that inhibition of cellular tyrosine kinase activity suppressed UVB-dependent effects in human skin. We confirmed that the same suppression of UVB-induced apoptosis occurs in normal human keratinocytes grown in culture. Furthermore, we sought to determine the role of erbB receptor tyrosine kinases in human keratinocytes following UVB irradiation. Using a specific inhibitor of the erbB family of tyrosine kinase receptors, DAPH, we investigated the effects of UVB-dependent activation of these receptors on keratinocyte biology. The addition of DAPH to keratinocytes resulted in the concentration-dependent protection of UVB-induced apoptosis. The protection from apoptosis was not due to the induction of keratinocyte differentiation, the loss of keratinocyte viability, or inhibition of the proliferative potential of keratinocytes by DAPH. The effect of DAPH on apoptosis was specific for UVB as it had no effect on bleomycin-induced apoptosis. Furthermore, the inhibition of UVB-induced apoptosis could also be observed using neutralizing antibodies to either erbB1 or erbB2. Finally, we demonstrated that DAPH could also inhibit UVB-induced apoptosis in an epidermal organotypic model system. These studies suggest an important role for the erbB receptors in UVB-induced apoptosis of human keratinocytes.

Fibroblast Senescence and Squamous Cell Carcinoma: How Wounding Therapies Could Be Protective

BACKGROUND Squamous cell carcinoma (SCC), which has one of the highest incidences of all cancers in the United States, is an age‐dependent disease, with the majority of these cancers diagnosed in people age 70 and older. Recent findings have led to a new hypothesis on the pathogenesis of SCC. OBJECTIVES To evaluate the potential of preventive therapies to reduce the incidence of SCC in at‐risk geriatric patients. MATERIALS AND METHODS Survey of current literature on wounding therapies to prevent SCCs. RESULTS This new hypothesis of SCC photocarcinogenesis states that senescent fibroblasts accumulate in the dermis, resulting in a reduction in dermal insulin‐like growth factor‐1 (IGF‐1) expression. This lack of IGF‐1 expression sensitizes epidermal keratinocytes to fail to suppress ultraviolet light B (UVB)‐induced mutations, leading to increased proclivity to photocarcinogenesis. Recent evidence suggests that dermal wounding therapies, specifically dermabrasion and fractionated laser resurfacing, can decrease the proportion of senescent dermal fibroblasts, increase dermal IGF‐1 expression, and correct the inappropriate UVB response found in geriatric skin, protecting geriatric keratinocytes from UVB‐induced SCC initiation. CONCLUSIONS In this review, we will discuss the translation of pioneering basic science results implicating commonly used dermal fibroblast rejuvenation procedures as preventative treatments for SCC.

A Radiation-Induced Acute Apoptosis Involving TP53 and BAX Precedes the Delayed Apoptosis and Neoplastic Transformation of CGL1 Human Hybrid Cells

Abstract Mendonca, M. S., Mayhugh, B. M., McDowell, B., Chin-Sinex, H., Smith, M. L., Dynlacht, J. R., Spandau, D. F. and Lewis, D. A. A Radiation-Induced Acute Apoptosis Involving TP53 and BAX Precedes the Delayed Apoptosis and Neoplastic Transformation of CGL1 Human Hybrid Cells. Radiat. Res. 163, 614–622 (2005). Exposing CGL1 (HeLa × fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of ∼50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-XL is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.

Insulin-like growth factor-1 receptor regulates repair of ultraviolet B-induced DNA damage in human keratinocytes in vivo

The activation status of the insulin‐like growth factor‐1 receptor (IGF‐1R) regulates the cellular response of keratinocytes to ultraviolet B (UVB) exposure, both in vitro and in vivo. Geriatric skin is deficient in IGF‐1 expression resulting in an aberrant IGF‐1R‐dependent UVB response which contributes to the development of aging‐associated squamous cell carcinoma. Furthermore, our lab and others have reported that geriatric keratinocytes repair UVB‐induced DNA damage less efficiently than young adult keratinocytes. Here, we show that IGF‐1R activation influences DNA damage repair in UVB‐irradiated keratinocytes. Specifically, in the absence of IGF‐1R activation, the rate of DNA damage repair following UVB‐irradiation was significantly slowed (using immortalized human keratinocytes) or inhibited (using primary human keratinocytes). Furthermore, inhibition of IGF‐1R activity in human skin, using either ex vivo explant cultures or in vivo xenograft models, suppressed DNA damage repair. Primary keratinocytes with an inactivated IGF‐1R also exhibited lower steady‐state levels of nucleotide excision repair mRNAs. These results suggest that deficient UVB‐induced DNA repair in geriatric keratinocytes is due in part to silenced IGF‐1R activation in geriatric skin and provide a mechanism for how the IGF‐1 pathway plays a role in the initiation of squamous cell carcinoma in geriatric patients.