UVB‐generated Microvesicle Particles: A Novel Pathway by Which a Skin‐specific Stimulus Could Exert Systemic Effects

Ultraviolet B radiation (UVB) exerts profound effects on human skin. Much is known regarding the ability of UVB to generate a plethora of bioactive agents ranging from cytokines and other bioactive proteins, lipid mediators and microRNAs. It is presumed that these agents are in large part responsible for the effects of UVB, which is only absorbed appreciably in the epidermis. However, the exact mechanism by which these bioactive agents can leave the epidermis are as yet unclear. This review addresses the potential role of microvesicle particles (MVP) as UVB signaling agents through transmitting biologic mediators. New data are provided that UVB treatment of human skin explants also generates MVP production. We hypothesize that UVB production of MVPs (UVB‐MVP) could serve this important function of transmitting keratinocyte‐derived bioactive agents. Moreover, we propose that UVB‐MVP formation involves the lipid mediator platelet‐activating factor. This novel pathway has the potential to be exploited pharmacologically to modulate UVB effects.     

Epidermal Platelet-activating Factor Receptor Activation and Ultraviolet B Radiation Result in Synergistic Tumor Necrosis Factor-alpha Production

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production which has been implicated in photoaggravated dermatoses. In addition to cytokines such as tumor necrosis factor-α (TNF-α), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous studies have demonstrated that UVB-mediated production of keratinocyte TNF-α is in part due to PAF. The current studies use a human PAF-receptor (PAF-R) negative epithelial cell line transduced with PAF-Rs and PAF–R-deficient mice to demonstrate that activation of the epidermal PAF-R along with UVB irradiation results in a synergistic production of TNF-α. It should be noted that PAF-R effects are mimicked by the protein kinase C (PKC) agonist phorbol myristic acetate, and are inhibited by pharmacological antagonists of the PKC gamma isoenzyme. These studies suggest that concomitant PAF-R activation and UVB irradiation results in a synergistic production of the cytokine TNF-α which is mediated in part via PKC. These studies provide a novel potential mechanism for photosensitivity responses.     

Plantamajoside Inhibits UVB and Advanced Glycation End Products‐Induced MMP‐1 Expression by Suppressing the MAPK and NF‐κB Pathways in HaCaT Cells

Photoaging and glycation stress are major causes of skin deterioration. Oxidative stress caused by ultraviolet B (UVB) irradiation can upregulate matrix metalloprotease 1 (MMP‐1), a major enzyme responsible for collagen damage in the skin. Advanced glycation end products (AGEs) accumulate via gradual formation from skin proteins, especially from long‐lived proteins such as dermal elastin and collagen. Plantamajoside (PM), isolated from Plantago asiatica, has various biological effects including anti‐inflammatory and antioxidant effects. In this study, we assessed the protective effects of PM on a human keratinocyte cell line (HaCaT) and primary human dermal fibroblasts (HDF) against stress caused by glyceraldehyde‐induced AGEs (glycer‐AGEs) with UVB irradiation. We found that PM attenuated UVB‐ and‐glycer‐AGEs‐induced MMP‐1 expression in HaCaT and HDF cells and proinflammatory cytokines expression by inhibiting the phosphorylation of mitogen‐activated protein kinases (MAPKs) activated by reactive oxygen species. Specific inhibitors of NF‐κB and MAPKs attenuated the induced expression of MMP‐1. PM also inhibited the phosphorylation of IκBα, and reduced nuclear translocation of NF‐κB in these cells. Furthermore, PM attenuated the upregulation of receptor for AGEs (RAGE) by glycer‐AGEs with UVB irradiation. Therefore, our findings strongly suggest that PM is a promising inhibitor of skin photoaging.     

Requirement for Metalloproteinase‐dependent ERK and AKT Activation in UVB‐induced G1‐S Cell Cycle Progression of Human Keratinocytes

UVB (280–315 nm) in natural sunlight represents a major environmental challenge to the skin and is clearly associated with human skin cancer. Here we demonstrate that low doses of UVB induce keratinocyte proliferation and cell cycle progression of human HaCaT keratinocytes. Different from UVA, UVB irradiation induced extracellular signal‐regulated kinase (ERK) and AKT activation and their activation are both required for UVB‐induced cell cycle progression. Activation of epidermal growth factor receptor (EGFR) was observed after UVB exposure and is upstream of ERK/AKT/cyclin D1 pathway activation and cell cycle progression following UVB radiation. Furthermore, metalloproteinase (MP) inhibitor GM6001 blocked UVB‐induced ERK and AKT activation, cell cycle progression, and decreased the EGFR phosphorylation, demonstrating that MPs mediate the EGFR/ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB radiation. In addition, ERK or AKT activation is essential for EGFR activation because ERK or AKT inhibitor blocks EGFR activation following UVB radiation, indicating that EGFR/AKT/ERK pathways form a regulatory loop and converge into cell cycle progression following UVB radiation. Identification of these signaling pathways in UVB‐induced cell cycle progression of quiescent keratinocytes as a process mimicking tumor promotion in vivo will facilitate the development of efficient and safe chemopreventive and therapeutic strategies for skin cancer.     

Ultraviolet B Radiation of Human Skin Generates Platelet-activating Factor Receptor Agonists

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production. In addition to cytokines, such as tumor necrosis factor-alpha (TNF-α), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous in vitro studies in keratinocytes or epithelial cell lines have demonstrated that UVB-mediated production of PAF agonists is due primarily to the pro-oxidative effects of this stimulant, resulting in the nonenzymatic production of modified phosphocholines (oxidized glycerophosphocholines). The current studies use human skin to assess whether UVB irradiation generates PAF-receptor agonists, and the role of oxidative stress in their production. These studies demonstrate that UVB irradiation of human skin results in PAF agonists, which are blocked by the antioxidant vitamin C and the epidermal growth factor receptor inhibitor PD168393. Inasmuch as UVB-generated PAF agonists have been implicated in animal model systems as being involved in photobiologic processes including systemic immunosuppression and cytokine (TNF-α) production, these studies indicate that this novel activity could be involved in human disease.     

Research Note: Ultraviolet Irradiation (UVB) Interrupts Calcium Cell Signaling in Lens Epithelial Cells

A preliminary study was undertaken to establish whether low-dose UV irradiation (UVB) affects calcium cell signaling in rabbit lens epithelia. In a suspension of lens epithelial cells (line NN1003A), changes in intracellular Ca2+ were measured by Fura-2 fluorescence in response to exogenously added ATP. The cellular response to ATP, referred to as the calcium signal, is characterized by a brief increase and subsequent decrease in cytosolic Ca2+ levels. Ultraviolet B irradiation (1.8-9 mJ/cm2) was found to reduce the magnitude of the Ca2+ signal in a dose-dependent manner. A 5 min UVB exposure (9 mJ/cm2) completely altered the biphasic nature of the calcium signal, causing only an immediate and steady rise in cytosol Ca2+ levels. Lower fluences of UVB irradiation (2 min exposure times or 3.6 mJ/cm2) induced a 50% reduction in the calcium signal. When irradiated cells were returned to culture for 3 h after irradiation, calcium signals induced by ATP were normal. In view of the photooxidative nature of UVB irradiation, the oxidative state of cells was assessed by measuring glutathione (GSH) levels. Ultraviolet B irradiation caused a rapid 20% decline in GSH levels that returned to near-control values after a 3 h postirradiation incubation. The results of this study indicate that fluences lower than previously found to be cataractogenic can perturb calcium cell signaling in cultured lens epithelial cells.     

UVB Irradiation Enhances TiO2 Nanoparticle‐induced Disruption of Calcium Homeostasis in Human Lens Epithelial Cells

Currently, titanium dioxide nanoparticles (TiO₂ NPs) have been widely used in various applications including cosmetics, food additives and biomedicine. However, there are few reports available using TiO₂ NPs to treat ocular diseases. Posterior capsular opacification (PCO) is the most frequent complication after cataract surgery, which is induced by the proliferation and migration of lens epithelial cells. Thus, inhibiting the proliferation of lens epithelial cells will efficiently reduce the occurrence of PCO. In this study, we investigated the effects of TiO₂ NPs on HLE B‐3 cells with or without ultraviolet B (UVB) irradiation in vitro. We found that TiO₂ NPs can inhibit HLE B‐3 cell growth, cause the elevation of intracellular [Ca²⁺], produce excessive reactive oxygen species (ROS), further reduce Ca²⁺‐ATPase activity and decrease the expression of plasma membrane calcium ATPase 1 (PMCA1), finally disrupt the intracellular calcium homeostasis and induce cell damage. Importantly, UVB irradiation can apparently enhance these effects on HLE B‐3 cells in the presence of TiO₂ NPs. Taken together, the generation of excessive ROS and the disruption of intracellular calcium homeostasis may be both involved in TiO₂ nanoparticle‐induced HLE B‐3 cell damage under UVB irradiation.     

The role of cholesterol in UV light B-induced apoptosis

Modification of major lipid raft components, such as cholesterol and ceramide, plays a role in regulation of programmed cell death under various stimuli. However, the relationship between cholesterol level modification and the activation of apoptotic signaling cascades upon UVB light has not been established. In this report, we demonstrate that upon UVB irradiation cholesterol levels in membrane rafts of skin cells increase, which leads to Fas-receptor (Fas) aggregation in the rafts. Utilizing a continuous velocity floatation technique, we show that Fas accumulated in the lipid rafts of human melanoma M624 cells after UVB irradiation. The subsequent events of death-inducing signaling complex formation were also detected in the lipid raft fractions. Depletion of cholesterol by methyl-β-cyclodextrin reduces Fas aggregation, while overloading increases. Disruption of lipid rafts also prevents Fas death domain-associated protein (Daxx) from dissociating from Fas in the lipid rafts, which is accompanied with a reduced apoptotic, but increased nonapoptotic death of UVB-irradiated human keratinocytes, HaCaT cells. Results indicate that cholesterol located in the plasma membrane of skin cells is required for lipid raft domain formation and activation of UVB-induced apoptosis.     

Protective Effect of Curcumin Against Acute Ultraviolet B Irradiation‐induced Photo‐damage

Ultraviolet B (UVB) irradiation is one of the most dangerous insults for skin and causes sunburn, erythema, photoaging and photocarcinogenesis. Curcumin (diferuloylmethane), a yellow spice derived from dried rhizomes of Curcuma longa, has been shown to possess significant anti‐inflammatory, antioxidant, anticarcinogenic, antimutagenic, anticoagulant and anti‐infective effects. However, the protective effects of curcumin against acute photo‐damage are poorly understood. In this study, we investigated the photoprotective effects of curcumin against UVB‐induced acute photo‐damage in hairless mice and immortalized human keratinocytes (HaCaT). Topical application of curcumin significantly inhibited acute UVB (540 mJ cm^?2, for 3 successive days)‐induced inflammatory cells, collagen accrementition derangement and lipid peroxidation, and effectively induced NF‐E2‐related factor 2 (Nrf2) nuclear accumulation in uncovered (Uncv) hairless mice skin. Treatment of HaCaT cells with curcumin significantly attenuated acute UVB (300 mJ cm^?2)‐induced lactate dehydrogenase release, intracellular reactive oxygen species production and DNA damage, activated the expression of the phase II detoxifying enzymes and promoted DNA repair activity. The photoprotective effect provided by curcumin was potential associated with modulation of Nrf2‐dependent antioxidant response. Our study suggested that curcumin is a potential agent for preventing and/or treating UV radiation‐induced acute inflammation and photoaging.     

胶内差异双向电泳-电喷雾串联质谱筛选UVB损伤皮肤细胞潜在的蛋白质靶标

联用胶内差异双向电泳(2D-DIGE)和高效液相色谱-电喷雾串联质谱(HPLC-nESI MS/MS)鉴定人角质形成细胞HaCaT应答中波紫外线(UVB)损伤的差异表达蛋白,筛选UVB影响皮肤细胞正常生理功能潜在的靶标蛋白.结果表明:UVB辐射明显影响HaCaT细胞的蛋白质表达谱,DeCyder软件在每块DIGE凝胶上...     

Induction of Proinflammatory Cytokines in Human Epidermoid Carcinoma Cells by in vitro Ultraviolet A1 Irradiation

Abstract— Ultraviolet radiation-induced expression of cytokines by keratinocytes is important for the pathogenesis of polymorphous light eruption (PLE). Because UVA1 radiation rather than UVB radiation might be a more important trigger for PLE, cells from the human epidermoid carcinoma cell line KB were exposed in vitro to UVA1 radiation (30 J/cm²) and subsequently analyzed for cytokine expression. Ultraviolet A1 irradiation induced tumor necrosis factor (TNF)-a and interleukin (IL)-8 expression in KB cells at the mRNA and protein level. Upregulation of cytokine mRNA levels followed a Diphasic pattern. This effect was specific for TNFa and IL-8 because UVA1 radiation did not induce expression of IL-la or IL-6 in these cells. Ultraviolet Al radiation-induced expression of intercellular adhesion molecule-1 in KB cells previously was found to depend on the thiol status of these cells. Therefore, KB cells were treated with DL-buthionine-[S, R]-sulfoximine (BSO), a specific inhibitor of de novo glutathione synthesis. Exposure of BSO-pretreated KB cells to UVA1 radiation significantly induced IL-1α and IL-6 mRNA and protein expression. These studies demonstrate the capacity of UVA1 radiation to induce cytokine expression in human epidermoid carcinoma cells. This immunomodulatory effect may be mediated by thiol-status-dependent and -independent mechanisms.     

Platelet-activating factor does not mediate UVB-induced local immune suppression

The lipid mediator Platelet-activating factor (PAF) and oxidized glycerophosphocholine PAF agonists produced by UVB have been demonstrated to play a pivotal role in UVB-mediated systemic immunosuppression. Importantly, employing the ability of distant UVB irradiation to inhibit contact hypersensitivity (CHS) responses to the chemical antigen dinitrofluorobenzene (DNFB) to an area of unirradiated murine skin, we and others have demonstrated that UVB-mediated systemic immunosuppression was only observed in PAF-R expressing wild type (WT) mice and not in PAF-R-knockout (Pafr-/-) mice. As it is not known if PAF is involved in UVB-mediated local immunosuppression, these studies compared local UVB on CHS responses in WT versus Pafr-/- mice. We demonstrate that the application of DNFB onto UVB-exposed (locally) area of mouse skin resulted in a similar significant inhibition of subsequent CHS responses in both WT and Pafr-/- mice compared to sham-irradiated control mice. Furthermore, the expression of langerin, a marker for the presence of Langerhans cells was substantially reduced equally in the epidermal ears of UVB-irradiated WT and Pafr-/- mice compared to their respective sham control groups. These findings indicate that the PAF-R is not involved UVB-induced local immunosuppression.     

Narrow‐band UVB‐induced Externalization of Selected Nuclear Antigens in Keratinocytes: Implications for Lupus Erythematosus Pathogenesis†

The aim of this study was to analyze whether sera obtained from patients with lupus erythematosus (LE) react with membrane structures found on keratinocytes irradiated with narrow‐band ultraviolet B (NB‐UVB). We applied atomic force microscopy (AFM) to visualize cell surface structures expressing nuclear antigens upon apoptosis following NB‐UVB irradiation. Immortalized human keratinocytes (HaCaT) were cultured under standard conditions, irradiated with 800 mJ cm^?2 NB‐UVB light and imaged by AFM mounted on an inverted optical microscope. It was observed that NB‐UVB irradiation provoked significant alterations of the keratinocyte morphology and led to the membrane expression of antigens recognized by anti‐La and anti‐Ro 60 kDa sera but not by antidouble‐strand DNA sera. The presence of La and Ro 60 kDa antigens on keratinocyte surfaces after NB‐UVB irradiation was limited mainly to the small bleb‐like protrusions found on the keratinocytes by AFM. A closer investigation by AFM also revealed that some structures positively stained with anti‐Ro 60 kDa serum were also located submembranously. We hypothesize that the externalization of some nuclear antigens because of NB‐UVB exposure might be responsible for exacerbation of skin symptoms in patients suffering from LE.     

Bucillamine Inhibits UVB-Induced MAPK Activation and Apoptosis in Human HaCaT Keratinocytes and SKH-1 Hairless Mouse Skin

Ultraviolet B (UVB) radiation is known as a culprit in skin carcinogenesis. We have previously reported that bucillamine (N-[2-mercapto-2-methylpropionyl]-L-cysteine), a cysteine derivative with antioxidant and anti-inflammatory capacity, protects against UVB-induced p53 activation and inflammatory responses in mouse skin. Since MAPK signaling pathways regulate p53 expression and activation, here we determined bucillamine effect on UVB-mediated MAPK activation in vitro using human skin keratinocyte cell line HaCaT and in vivo using SKH-1 hairless mouse skin. A single low dose of UVB (30 mJ cm⁻²) resulted in increased JNK/MAPK phosphorylation and caspase-3 cleavage in HaCaT cells. However, JNK activation and casaspe-3 cleavage were inhibited by pretreatment of HaCaT cells with physiological doses of bucillamine (25 and 100 µm ). Consistent with these results, bucillamine pretreatment in mice (20 mg kg⁻¹) inhibited JNK/MAPK and ERK/MAPK activation in skin epidermal cells at 6–12 and 24 h, respectively, after UVB exposure. Moreover, bucillamine attenuated UVB-induced Ki-67-positive cells and cleaved caspase-3-positive cells in mouse skin. These findings demonstrate that bucillamine inhibits UVB-induced MAPK signaling, cell proliferation and apoptosis. Together with our previous report, we provide evidence that bucillamine has a photoprotective effect against UV exposure.     

Expression and Function of Macrophage Migration Inhibitory Factor in the Pathogenesis of UV-Induced Cutaneous Nonmelanoma Skin Cancer(†)

Chronic skin exposure to ultraviolet light stimulates the production of cytokines known to be involved in the initiation of skin cancer. Recent studies in mouse models suggested a role for macrophage migration inhibitory factor (MIF) in the UVB‐induced pathogenesis of nonmelanoma skin cancer (NMSC). Our studies aimed at defining the pathophysiological function of MIF in cutaneous inflammatory reactions and in the development and progression of NMSC. Immunohistochemical analysis revealed a moderate expression of MIF in normal human skin samples but an enhanced expression of this cytokine in lesional skin of patients with actinic keratosis or cutaneous SCC. Enzyme‐linked immunosorbent assay studies showed a time‐dependent increase in MIF secretion after a moderate single‐dose UVB irradiation in NHEKs and SCC tumor cells. MIF is known to interact with CXCR2, CXCR4 and CD74. These receptors are not constitutively expressed in keratinocytes and HaCaT cells and their expression is not induced by UVB irradiation either. However, stimulation with IFNγ upregulated CD74 surface expression in these cells. Affymetrix^® Gene Chip analysis revealed that only keratinocytes prestimulated with IFNγ are responsive to MIF. These findings indicate that MIF may be an important factor in the pathogenesis of NMSC tumorigenesis and progression in an inflammatory environment.     

A Peptide YGDEY from Tilapia Gelatin Hydrolysates Inhibits UVB‐mediated Skin Photoaging by Regulating MMP‐1 and MMP‐9 Expression in HaCaT Cells

In this study, we investigated the protective effects of a peptide (YGDEY, Tyr‐Gly‐Asp‐Glu‐Tyr) isolated from tilapia skin gelatin hydrolysates (TGHs), against UVB‐induced photoaging in human keratinocytes (HaCaT) cells. Results showed that YGDEY significantly decreased levels of intracellular reactive oxygen species (ROS), increased antioxidant factors (Superoxide Dismutase, SOD and Glutathione, GSH) expression and maintained balance between GSH and GSSG in HaCaT cells. Comet assay shows that YGDEY can protect DNA from oxidative damage. Furthermore, it significantly inhibited MMP‐1 (collagenase) and MMP‐9 (gelatinase) expression and increased Type I procollagen production. In addition, the molecular docking study showed that YGDEY may form active sites with MMP‐1 and MMP‐9. Moreover, Western blot analysis was utilized to measure the protein levels of UVB‐induced mitogen‐activated protein kinase (MAPK) and nuclear factor‐kappa B (NF‐κB) signaling pathways. Therefore, these results suggested that YGDEY has a therapeutic effectiveness in prevention of UVB‐induced cellular damage, and it is a candidate worthy of being developed as a potential natural antioxidant and food additive.     

Influence of UVB,UVA and UVA1 irradiation on histamine release from human basophils and mast cells in vitro in the presence and absence of antioxidants

UV irradiation is widely used for the treatment of atopic eczema. In recent years, UVA1 phototherapy has gained increasing attention. This study analyzed the influence of different UV wavelengths--especially UVA1--on histamine release from human basophils and mast cells. The modulation of this parameter might be responsible for some of the therapeutic effects of UV irradiation. Enriched human basophils and human mast cells (HMC1 cell line) were irradiated with increasing doses of UVB, UVA and UVA1 in vitro. After irradiation, different stimulants were added to induce histamine release. In additional experiments, basophils were preincubated with superoxide dismutase, ascorbate or trolox to study the role of antioxidants in the modulation of histamine release after UV irradiation. UVA and UVA1 significantly inhibited histamine release from basophils and mast cells. UVB only had an inhibitory effect on mast cells. Preincubation with superoxide dismutase and ascorbate did not influence the inhibitory effect of UVA1 on basophil histamine release, whereas trolox decreased significantly the histamine release from nonirradiated basophils.     

The Effect of Endothelial Cells on UVB‐induced DNA Damage and Transformation of Keratinocytes In 3D Polycaprolactone Scaffold Co‐culture System

Nitric oxide ( ${\mathbf{NO}}^{·}$) plays an important role in the regulation of redox balance in keratinocytes post‐UVB exposure. Since endothelial cells releases ${\mathbf{NO}}^{·}$ for a prolonged time post‐UVB, we determined whether human umbilical vein endothelial cells (HUVEC) could have an effect on UVB‐induced DNA damage and transformation of their adjacent keratinocytes (HaCaT) using a 3D cell co‐culturing system. Our data show that the levels of DNA breaks and/or cyclobutane pyrimidine dimer (CPD) along with γH2AX are higher in the co‐cultured than in the mono‐cultured keratinocytes post‐UVB. The ${\mathbf{NO}}^{·}$ level in the co‐cultured cells is increased approximately 3‐fold more than in mono‐cultured HaCaT cells within 1‐hour post‐UVB but then is reduced quickly in co‐cultured HaCaT cells comparing to mono‐cultured cells from 6 to 24 h post‐UVB. However, the peroxynitrite (ONOO^?) level is higher in the co‐cultured than in the mono‐cultured HaCaT cells in whole period post‐UVB. Furthermore, while expression level of inducible nitric oxide synthase (iNOS) is increased, the ratio of coupled/uncoupled eNOS is reduced in co‐cultured HaCaT cells compared to mono‐cultured HaCaT cells. Finally, the co‐cultured cells have a significantly increased transformation efficiency after repeating UVB exposure compared to mono‐culture HaCaT cells. Our results suggest that endothelial cells could enhance ${\mathbf{NO}}^{·}$/ONOO^? imbalance and promote transformation of adjacent keratinocytes.     

UVA and UVB Radiation Differentially Regulate Vascular Endothelial Growth Factor Expression in Keratinocyte-derived Cell Lines and in Human Keratinocytes

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC-derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2 and 20 J/cm2) and UVB (8 mJ/cm2 and 16 mJ/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC-derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2 led to a marked decrease in soluble VEGF, whereas doses as high as 32 mJ/cm2 UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC-derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.