Tumoural angiogenesis is essential for the <em>growth</em> and spread of breast cancer cells. Therefore the aim of this study was to assess the diagnostic performance of angiogenesis markers in tumours and there reflecting levels in serum of breast cancer patients. Angiogenin, Ang<em>2</em>, fibroblast <em>growth</em> <em>factor</em> basic, intercellular adhesion molecule (ICAM)-1, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), platelet-derived <em>growth</em> <em>factor</em>-BB, and VEGF-A were measured using a FASTQuant angiogenic <em>growth</em> <em>factor</em> multiplex protein assay. We observed that breast cancer tumours exhibited high levels of PDGF-BB, bFGF and VEGF, and extremely high levels of TIMP-1 and Ang-<em>2</em>, whereas in serum we found significantly higher levels of Ang-<em>2</em>, PDGF-BB, bFGF, ICAM-1 and VEGF in patients with breast cancer compared to the benign breast diseases patients. Moreover, some of these angiogenesis markers evaluated in tumour and serum of breast cancer patients exhibited association with standard clinical parameters, ER status as well as MVD of tumours. Angiogenesis markers play important roles in tumour <em>growth</em>, invasion and metastasis. Our results suggest that analysis of angiogenesis markers in tumour and serum of breast cancer patients using multiplex protein assay can improve diagnosis and prognosis in this diseases.

We previously immortalized normal human oral <em>keratinocytes</em> by transfection with recombinant HPV-16 DNA and subsequently exposed the cells to benzo(a)pyrene for 7 days. The exposure to benzo(a)pyrene modified the immortalized cells: the modified cells (HOK-16B-BaP) proliferated in an ordinary culture medium containing physiological calcium level (1.5 mM), but demonstrated only enhanced proliferation capacity without tumor formation in nude mice and failed to show in vitro anchorage-independency. In this study, we further modified the HOK-16B-BaP cells by subculturing the cells in a medium containing benzo(a)pyrene for 6 months. The cells were further modified with a chronic benzo(a)pyrene exposure and were termed HOK-16B-BaP-T cells (1) demonstrated a malignant phenotype in organotypic 'raft' culture, (<em>2</em>) showed in vitro anchorage-independency, (3) developed tumors in nude mice when injected subcutaneously, (4) contained a significantly higher copy number of intact and integrated HPV-16 DNA; (5) contained higher level of HPV-16 E6/E7 messages and E7 protein, (6) were more resistant to transforming <em>growth</em> <em>factor</em>-beta 1 and (7) secreted higher level of vascular endothelial <em>growth</em> <em>factor</em> with molecular weight of 56 kd than parental HOK-16B-BaP cells. However, the levels of p53 and ras proteins and the levels of p53, c-myc and c-fos transcripts in the HOK-16B-BaP-T cells were not different from those in the HOK-16B-BaP cells. The highly conserved coding regions of the p53, c-Ha-ras1, and c-Ki-ras<em>2</em> genes of the tumor cells were not mutated. These data indicate that the HPV-immortalized human oral <em>keratinocytes</em> can convert to tumorigenic cells by chronic exposure to benzo(a)pyrene. The tumorigenic conversion seems to be associated with (1) the overexpression of viral oncogenes such as E6 and E7 genes, (<em>2</em>) the higher resistance of cells to transforming <em>growth</em> <em>factor</em>-beta 1 and (3) the high secretion of 56 kd vascular endothelial <em>growth</em> <em>factor</em> from the cells.

Rapidly <em>growing</em> normal human neonatal pro<em>keratinocyte</em>s (HPK) cultured in serum-free medium can be induced to undergo either reversible or irreversible <em>growth</em> arrest at distinct cell cycle states. Reversible G1 arrest was induced by culture of low-density cells in human lymphocyte conditioned medium, by culture in high-density stationary phase conditioned medium, and by culture in isoleucine-deficient medium. Irreversible arrest of HPK <em>growth</em> predominantly in G1 was induced by culture in <em>growth</em> <em>factor</em>-deficient medium. Irreversible arrest of HPK <em>growth</em> in G1 and G<em>2</em> was also induced by culture in suspension in methylcellulose prepared in complete MCDB 153 medium or by culture in serum-containing medium. Finally, the drug razoxane was employed to induce irreversible arrest of HPK in G<em>2</em>. These data establish that there are <em>2</em> distinct classes of <em>growth</em> arrest states for HPK and suggest that each arrest mechanism may serve a unique role in the control of <em>keratinocyte</em> differentiation in normal cells. It is also possible that the development of selective defects in either of these processes could be of etiologic significance in certain epidermal disease states.

Exposure of cultured human epidermal <em>keratinocytes</em> to the protein kinase C (Ca<em>2</em>+- and phospholipid-dependent protein kinase)-activating phorbol esters 1<em>2</em>-O-tetradecanoylphorbol-13-acetate (TPA) or 4-beta-phorbol-1<em>2</em>,13-didecanoate markedly enhanced accumulation of transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) mRNA and secretion of TGF-alpha protein. The nonactivating phorbol ester, 4-alpha-phorbol 1<em>2</em>,13-didecanoate, had no effect. In the absence of exogenous <em>growth</em> <em>factors</em>, confluent cultures of <em>keratinocytes</em> express low or undetectable levels of TGF-alpha mRNA and protein. While TPA and epidermal <em>growth</em> <em>factor</em> treatment of <em>keratinocyte</em> cultures deprived of <em>growth</em> <em>factors</em> both induced TGF-alpha mRNA expression, maximum induction by TPA is 5-fold greater than epidermal <em>growth</em> <em>factor</em>. Furthermore, the addition of epidermal <em>growth</em> <em>factor</em> did not enhance TPA-mediated induction of TGF-alpha mRNA expression. Under these experimental conditions, TPA increased levels of secreted TGF-alpha protein by <em>2</em>0-fold at <em>2</em>4 h. Concentration dependence and kinetic studies of TGF-alpha expression showed that TPA (greater than or equal to 1 ng/ml) induced accumulation of TGF-alpha mRNA with an optimum concentration of 10 ng/ml. TGF-alpha mRNA expression increased within 1 h following TPA treatment (10 ng/ml) and peaked at 5 h. At <em>2</em>4 h, TPA-treated cultures still expressed elevated levels of TGF-alpha mRNA (1.7-fold). Protein secretion into the medium was enhanced <em>2</em>-fold (5 h) to 3-fold (<em>2</em>4 h) by TPA treatment of <em>keratinocyte</em> cultures containing <em>growth</em> <em>factors</em>. Prolonged pretreatment (<em>2</em>4 h) of <em>keratinocyte</em> cultures with TPA caused marked desensitization of TGF-alpha mRNA expression to repeated stimulation by phorbol ester. The synthetic diacylglycerol, 1,<em>2</em>-sn-dioctanoylglycerol, enhanced levels of TGF-alpha transcription and secretion of TGF-alpha protein. The rate of TGF-alpha mRNA accumulation peaked and declined earlier for 1,<em>2</em>-sn-dioctanoylglycerol compared to TPA. 1,<em>2</em>-sn-Dioctanoylglycerol (50 micrograms/ml) increased production and secretion of TGF-alpha protein, but less than TPA treatment. An inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-<em>2</em>-methylpiperazine, also inhibited 1,<em>2</em>-sn-dioctanoylglycerol-mediated accumulation of TGF-alpha mRNA. Cycloheximide failed to inhibit TGF-alpha mRNA expression induced by TPA and, when added alone to <em>keratinocyte</em> cultures, significantly enhanced TGF-alpha mRNA accumulation. Actinomycin D abrogated transcriptional activation of TGF-alpha mRNA by TPA. These studies suggest that activation of protein kinase C by active phorbol esters or diacylglycerols is responsible, at least in part, for TGF-alpha gene expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidermal <em>growth</em> <em>factor</em> (EGF) is a potent chemotactic and mitogenic <em>factor</em> for epidermal <em>keratinocytes</em>, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this <em>growth</em> <em>factor</em> are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal <em>keratinocytes</em> with EGF, but not with other <em>growth</em> <em>factors</em>, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-<em>2</em> (ELMO<em>2</em>), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO<em>2</em>, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

Pretreatment of mice with the hemopoietic <em>growth</em> <em>factor</em>, FMS-like tyrosine kinase 3 ligand (Flt3L), has been shown to increase monocyte-derived myeloid dendritic cells (DC) in lung parenchymal tissue, with possible implications for protective immunity to lung bacterial infections. However, whether Flt3L treatment improves lung innate immunity of mice to challenge with Streptococcus pneumoniae has not been investigated previously. Mice pretreated with Flt3L exhibited a peripheral monocytosis and a strongly expanded lung myeloid DC pool, but responded with a similar proinflammatory cytokine release (TNF-alpha, IL-6, <em>keratinocyte</em> derived cytokine, MIP-<em>2</em>, CCL<em>2</em>) and neutrophilic alveolitis upon infection with S. pneumoniae as did control mice with a normal lung DC pool. Unexpectedly, however, Flt3L-pretreated mice, but not control mice, infected with S. pneumoniae developed vasculitis and increased lung permeability by days <em>2</em>-3 postinfection, and florid pneumonia accompanied by sustained increased bacterial loads by days 3-4 postinfection. This was associated with an overall increased mortality of approximately 35% by day 4 after pneumococcal challenge. Application of anti-CCR<em>2</em> Ab MC<em>2</em>1 to block inflammatory monocyte-dependent lung mononuclear phagocyte mobilization significantly reduced the lung leakage, but not vasculitis in Flt3L-pretreated mice infected with S. pneumoniae, without affecting the intra-alveolar cytokine liberation or the concomitantly developing neutrophilic alveolitis. Together, the data demonstrate that previous Flt3L-induced lung DC accumulation is not protective in lung innate immunity to challenge with S. pneumoniae, and support the concept that CCR<em>2</em>-dependent mononuclear phagocyte as opposed to neutrophil recruitment contributes to increased lung leakage in Flt3L-pretreated mice challenged with S. pneumoniae.

Epidermal <em>growth</em> <em>factor</em> (EGF) along with several related peptide <em>growth</em> <em>factors</em> has been shown both in vivo and in vitro to accelerate events associated with epidermal wound repair. EGF and transforming <em>growth</em> <em>factor</em> alpha act by binding to a common EGF receptor tyrosine kinase thereby initiating a series of events which ultimately regulate cell proliferation. This study examined the immunohistochemical localization of EGF receptor (EGF-R) in burn wound margins, adjacent proliferating epithelium, and closely associated sweat ducts, sebaceous glands, and hair follicles. Tissue specimens removed during surgical debridement were obtained from full and partial thickness burn wounds in 3<em>2</em> patients with total body surface area burns ranging from <em>2</em> to 88%. In the early postburn period (days <em>2</em>-4), prominent staining for EGF-R was found in undifferentiated, marginal <em>keratinocytes</em>, adjacent proliferating, hypertrophic epithelium, and both marginal and nonmarginal hair follicles, sweat ducts, and sebaceous glands. During the late postburn period (days 5-16), EGF-R was depleted along leading epithelial margins; however, immunoreactive EGF-R remained intensely positive in the hypertrophic epithelium and all skin appendages. Increased detection of immunoreactive EGF-R and the presence of [1<em>2</em>5I]EGF binding in the hypertrophic epithelium correlated positively with proliferating cell nuclear antigen distributions. Thus, the presence of EGF-R in the appropriate <em>keratinocyte</em> populations suggests a functional role for this receptor during wound repair. Dynamic modulation in EGF receptor distribution during the temporal sequence of repair provides further evidence that an EGF/transforming <em>growth</em> <em>factor</em> alpha/EGF-R-mediated pathway is activated during human wound repair.

K-sam, also designated fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em>/BEK, was originally cloned from a stomach cancer cell line KATO-III. The gene is amplified and overexpressed preferentially in poorly differentiated types of stomach cancers. The major K-sam transcript in KATO-III cells encodes a receptor protein with a truncated carboxyl terminus and with a high-affinity binding site for <em>keratinocyte</em> <em>growth</em> <em>factor</em>. This truncated type is produced by an alternative splicing mechanism, and in normal tissues, the truncated type is far less prevalent than the untruncated form. The variant K-sam complementary DNA lacks tyrosine 769, which is a putative phospholipase C gamma 1 association site, and showed a higher transforming activity to NIH3T3 cells than the untruncated form, which is identical with the <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor.

The epidermis consists of a squamous epithelium continuously replenished by committed stem cells, which can either self-renew or differentiate. We demonstrated previously that E<em>2</em>F genes are differentially expressed in developing epidermis (Dagnino, L., Fry, C. J., Bartley, S. M., Farnham, P., Gallie, B. L., and Phillips, R. A. (1997) Cell <em>Growth</em> Differ. 8, 553-563). Thus, we hypothesized that various E<em>2</em>F proteins likely play distinct <em>growth</em> regulatory roles in the undifferentiated stem cells and in terminally differentiated <em>keratinocytes</em>. To further understand the function of E<em>2</em>F genes in epidermal morphogenesis, we have examined the expression, regulation, and protein-protein interactions of E<em>2</em>F <em>factors</em> in undifferentiated cultured murine primary <em>keratinocytes</em> or in cells induced to differentiate with Ca(<em>2</em>+) or BMP-6 (bone morphogenetic protein 6). We find similar patterns of E<em>2</em>F regulation with both differentiating agents and demonstrate a switch in expression from E<em>2</em>F-1, -<em>2</em>, and -3 in undifferentiated, proliferating cells to E<em>2</em>F-5 in terminally differentiated <em>keratinocytes</em>. Inhibition of <em>keratinocyte</em> proliferation by transforming <em>growth</em> <em>factor</em>-beta1 did not enhance E<em>2</em>F-5 protein levels, suggesting that this response is specific to differentiation rather than reversible cell cycle withdrawal. E<em>2</em>F-5 up-regulation is also accompanied by formation of heteromeric nuclear complexes containing E<em>2</em>F5, p130, and histone deacetylase (HDAC) 1. Overexpression of E<em>2</em>F5 specifically inhibited DNA synthesis in undifferentiated <em>keratinocytes</em> in an HDAC-dependent manner, suggesting that E<em>2</em>F-5.p130.HDAC1 complexes are likely involved in the permanent withdrawal from the cell cycle of <em>keratinocytes</em> responding to differentiation stimuli.

The cutaneous response to injury and stress comprises a temporary change in the balance between epidermal proliferation and differentiation as well as an activation of the immune system. Soluble <em>factors</em> play an important role in the regulation of these complex processes by coordinating the intercellular communication between <em>keratinocytes</em>, fibroblasts, and inflammatory cells. In this study, we demonstrate that JunB, a member of the activator protein-1 transcription <em>factor</em> family, is an important regulator of cytokine expression and thus critically involved in the cutaneous response to injury and stress. Mice lacking JunB in the skin develop normally, indicating that JunB is neither required for cutaneous organogenesis, nor homeostasis. However, upon wounding and treatment with the phorbol ester 1<em>2</em>-O-decanoyl-phorbol-13-acetate, JunB-deficiency in the skin likewise resulted in pronounced epidermal hyperproliferation, disturbed differentiation, and prolonged inflammation. Furthermore, delayed tissue remodelling was observed during wound healing. These phenotypic skin abnormalities were associated with JunB-dependent alterations in expression levels and kinetics of important mediators of wound repair, such as granulocyte macrophage colony-stimulating <em>factor</em>, <em>growth</em>-regulated protein-1, macrophage inflammatory protein-<em>2</em>, and lipocalin-<em>2</em> in both the dermal and epidermal compartment of the skin, and a reduced ability of wound contraction of mutant dermal fibroblasts in vitro.

Thiazolidinediones are a novel class of antidiabetic drugs that improve insulin sensitivity in type <em>2</em> diabetic patients. Recently, these compounds have also been shown to suppress tumor development in several animal models. The molecular basis for their antitumor action, however, is largely unknown. We report here that oral administration of thiazolidinediones (rosiglitazone and troglitazone) remarkably inhibited insulin-like <em>growth</em> <em>factor</em>-I (IGF-I)-promoted skin tumor development by 73% in BK5.IGF-1 transgenic mice, although they were previously found to be ineffective in inhibiting UV- or chemically induced mouse skin tumorigenesis. The anti-IGF-I effect of troglitazone in mouse skin <em>keratinocytes</em> was due to, at least partially, inhibition of IGF-I-induced phosphorylation of p70S6 kinase (p70S6K) at Thr(389), a site specifically phosphorylated by mammalian target of rapamycin (mTOR). Troglitazone did not directly inhibit mTOR kinase activity as shown by mTOR in vitro kinase assay but rapidly activated AMP-activated protein kinase (AMPK) through a yet undefined peroxisome proliferator-activated receptor gamma-independent mechanism. Expression of a dominant-negative AMPK reversed the inhibitory effect of troglitazone on IGF-I-induced phosphorylation of p70S6K, suggesting that troglitazone inhibited IGF-I and p70S6K signaling through activation of AMPK. Collectively, these data suggest that thiazolidinediones specifically inhibit IGF-I tumor-promoting activity in mouse skin through activation of AMPK and subsequent inhibition of p70S6K.

OBJECTIVE

The study aims to evaluate if human keratinocyte growth factor (hKGF), secreted after transduction of murine salivary glands with adenoviral vectors, can prevent oral mucositis resulting from radiation.

METHODS

Two serotype 5 adenoviral vectors encoding hKGF were constructed: AdEF1alpha-hKGF and AdLTR(2)EF1alpha-hKGF. Female C3H mice, 8 weeks old, were irradiated by single (22.5 Gy) or fractionated (5 x 8 Gy for 5 days) doses to induce oral mucositis (ulcers on tongue). One day before irradiation, the above viral vectors or an empty vector, Adcontrol, was given (10(10) particles per gland) to both submandibular glands by retrograde ductal instillation. Each experiment included five groups: no irradiation and irradiation (+/-Adcontrol, AdEF1alpha-hKGF, or AdLTR(2)EF1alpha-hKGF). Blood, saliva, submandibular glands, and tongue were collected on day 7 for single-dose studies or day 10 for fractionated dosing. hKGF levels were measured by ELISA.

RESULTS

In three separate single-dose irradiation experiments, lingual ulcers were dramatically reduced after either KGF-expressing vector. Similarly, in two separate fractionated irradiation experiments, the hKGF-expressing vectors completely prevented ulcer formation. QPCR data indicated that approximately 10(7) to 10(8) particles of each vector remained in the targeted submandibular glands at the terminal time. Transgenic hKGF protein was found at high levels in saliva, serum, and submandibular gland extracts.

CONCLUSIONS

hKGF gene transfer to salivary glands prevented radiation-induced oral mucositis in mice. This proof of concept study suggests that transgenic hKGF secreted from transduced salivary glands may be useful clinically to prevent oral mucositis caused by radiation.

The re-epithelialization of the wound involves the migration of <em>keratinocytes</em> from the edges of the wound. During this process, <em>keratinocyte</em> migration and proliferation will depend on the interaction of <em>keratinocytes</em> with dermal fibroblasts and the extracellular matrix. The present study aimed to investigate (1) the role of fibroblasts in the re-epithelialization process and on the reconstitution of the dermal-epidermal junction (DEJ) and (<em>2</em>) differential protein expression during re-epithelialization. For both purposes, three-dimensional human skin equivalents (HSE) were used. A full-thickness wound in HSE was introduced by freezing with liquid nitrogen and a superficial wound by linear incision with a scalpel. The closure of the wound in the absence or presence of exogenous <em>growth</em> <em>factors</em> was followed by monitoring the rate of re-epithelialization and regeneration of the DEJ. The results obtained in this study demonstrate that fibroblasts facilitate wound closure, but they differentially affected the deposition of various basement membrane components. The deposition of laminin 5 at the DEJ was delayed in superficial wounds as compared to the full-thickness wounds. During freeze injury, some basement membrane (BM) components remain associated with the dermal compartment and probably facilitate the BM reconstitution. The re-epithelialization process in full-thickness but not in superficial wounds was accelerated by the presence of <em>keratinocyte</em> <em>growth</em> <em>factor</em> and especially by epidermal <em>growth</em> <em>factor</em>. In addition, we have examined the deposition of various basement membrane components and the differences in protein expression in a laterally expanding epidermis in uninjured HSE. Laminin 5, type IV and VII collagen deposition was decreased in the laterally expanding epidermis, indicating that the presence of these proteins is not required for <em>keratinocyte</em> migration to occur in vitro. Using two-dimensional polyacrylamide gel electrophoresis, we have identified DJ-1, a protein not earlier reported to be differently expressed during the epithelialization process of the skin.

Interferon-gamma (IFN gamma) and 1,<em>2</em>5-dihydroxyvitamin D [1,<em>2</em>5-(OH)<em>2</em>D] each have potent antiproliferative and prodifferentiating effects on <em>keratinocytes</em>. Since <em>keratinocytes</em> produce 1,<em>2</em>5-(OH)<em>2</em>D, we explored the possibility that IFN gamma acted on <em>keratinocytes</em> in part by regulating 1,<em>2</em>5-(OH)<em>2</em>D production. We cultured human neonatal foreskin <em>keratinocytes</em> for various periods of time in the presence of various concentrations of IFN gamma before assessing their ability to produce 1,<em>2</em>5-(OH)<em>2</em>D. The production of 1,<em>2</em>5-(OH)<em>2</em>D by preconfluent <em>keratinocytes</em> grown in the presence of serum (which retards differentiation) was stimulated by 1.8 nM IFN gamma. Postconfluent <em>keratinocytes</em> did not respond to 1.8 nM IFN gamma. The production of 1,<em>2</em>5-(OH)<em>2</em>D by <em>keratinocytes</em> grown in serum-free medium was maximally stimulated by 0.006 nM IFN gamma and inhibited at concentrations greater than 0.06 nM. <em>Keratinocytes</em> grown in 0.1 mM calcium serum-free medium (which prevents differentiation) were more sensitive to both the stimulatory and inhibitory effects of IFN gamma than <em>keratinocytes</em> grown in 1.<em>2</em> mM calcium serum-free medium (which permits differentiation). The stimulatory effect of IFN gamma on 1,<em>2</em>5-(OH)<em>2</em>D production was maximal after <em>2</em> days of incubation. Incubations longer than <em>2</em> days showed increasingly less stimulation at the low IFN gamma concentrations and increasingly greater inhibition at the higher IFN gamma concentrations. The inhibitory effects of IFN gamma on 1,<em>2</em>5-(OH)<em>2</em>D production paralleled the inhibitory effects of IFN gamma on cell <em>growth</em>. Thus, IFN gamma does regulate 1,<em>2</em>5-(OH)<em>2</em>D production by <em>keratinocytes</em>. However, this regulation is modulated by the state of <em>keratinocyte</em> proliferation and differentiation and is influenced by calcium and undefined <em>factors</em> in serum. The data are consistent with the possibility that IFN gamma alters <em>keratinocyte</em> differentiation in part by regulating 1,<em>2</em>5-(OH)<em>2</em>D production.

Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in <em>keratinocytes</em>. Using the human <em>keratinocyte</em> cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 microM) of NO-donating agents ¿S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[<em>2</em>-(<em>2</em>-aminoethyl)-N-(<em>2</em>-ammonioethyl) amino] diazen-1-ium-1,<em>2</em> -diolate (DETA-NO)¿, but not by serum or by single recombinant <em>growth</em> <em>factors</em> and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in <em>keratinocytes</em>. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 microM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 microM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in <em>keratinocytes</em>, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on <em>keratinocyte</em> proliferation.

c-Myc overexpression has been associated with several types of human cancers. To study the role of c-myc in epidermal differentiation and carcinogenesis, a transgenic mouse model was created to overexpress c-Myc in the epidermis. Human c-myc <em>2</em> cDNA was subcloned into a 6.5 kb mouse loricrin expression vector, ML.myc<em>2</em>. This loricrin promoter primarily directs expression in the epidermis in both proliferating and differentiated <em>keratinocytes</em>. On day 4, ML.myc<em>2</em> transgenic pups develop a hyperkeratotic phenotype, which progressively worsens until day 7. Upon histological analysis, both hyperplasia and hyperkeratosis were evident. Bromodeoxyuridine (BrdU) incorporation revealed that transgenic mice had a threefold increase in the number of proliferating cells as compared with a normal littermate. Proliferative cells in the ML.myc<em>2</em> epidermis were also found to be suprabasal, suggesting an inhibition of terminal differentiation in <em>keratinocytes</em>. Inhibition of terminal differentiation by c-Myc overexpression was further suggested by aberrant expression of differentiation markers, keratin 1, keratin 6, loricrin, and filaggrin in ML.myc<em>2</em> transgenic mice. Interestingly, ML.myc<em>2</em> <em>keratinocytes</em> exhibit a reduced sensitivity to UV-B induced apoptosis, in vivo. In vitro studies reveal the reduced sensitivity of ML.myc<em>2</em> <em>keratinocytes</em> to UV-B irradiation is <em>growth</em> <em>factor</em> dependent. These findings provide evidence that overexpression of c-Myc in the epidermis induces proliferation, inhibits terminal differentiation and decreases the sensitivity of <em>keratinocytes</em> to UV-B induced apoptosis.

To elucidate biologic functions of hepatocyte <em>growth</em> <em>factor</em> and the c-Met receptor in cutaneous wound healing, we analyzed expression and localization of hepatocyte <em>growth</em> <em>factor</em> and c-Met receptor and used a strategy to neutralize endogenous hepatocyte <em>growth</em> <em>factor</em> in a cutaneous wound healing model in mice. Following excision of full-thickness skin on the dorsum of mice, expression of both hepatocyte <em>growth</em> <em>factor</em> and the c-Met receptor increased transiently in cutaneous tissues. Expressions of hepatocyte <em>growth</em> <em>factor</em> increased as early as <em>2</em> d postwounding and reached a peak on day <em>2</em>, whereas the c-Met receptor expression reached a peak <em>2</em>-4 d postwounding. Immunolocalization of the c-Met receptor indicated that c-Met receptor expression was upregulated in <em>keratinocytes</em>, vascular endothelial cells, and myofibroblasts in granulation tissue, hence these are potential target cells of hepatocyte <em>growth</em> <em>factor</em>. When normal rabbit IgG or neutralizing anti-hepatocyte <em>growth</em> <em>factor</em> IgG was locally and continuously delivered to subcutaneous lesions, the number of capillary vessels decreased with the neutralization of hepatocyte <em>growth</em> <em>factor</em> and there was an associated decreased expansion of granulation tissue. Likewise, retardation in re-epithelialization and the rate of wound closure occurred with neutralization of endogenous hepatocyte <em>growth</em> <em>factor</em> on days 4 and 7 postwounding. Therefore, hepatocyte <em>growth</em> <em>factor</em> is definitely involved in enhancing cutaneous wound healing processes, including re-epithelialization, neovascularization, and granulation tissue formation.

The effect of 1,<em>2</em>5-dihydroxyvitamin D3 [1,<em>2</em>5(OH)<em>2</em>D3] on the <em>growth</em> of normal human <em>keratinocytes</em> cultured in serum-free medium was investigated. 1,<em>2</em>5(OH)<em>2</em>D3 inhibited the cell <em>growth</em> at 10(-7) M by 75.3% and at 10(-6) M almost completely. The <em>growth</em> inhibition was accompanied by changes related to proliferation: (1) remarkable inhibition of DNA synthesis, (<em>2</em>) the decrease in the number of high-affinity receptors for epidermal <em>growth</em> <em>factor</em>, with almost no change in total receptor number, (3) the rapid decrease in c-myc mRNA level. The inhibition of DNA synthesis and the decrease of c-myc mRNA expression occurred at 3 h after the addition of 1,<em>2</em>5(OH)<em>2</em>D3. These results suggest that decrease of c-myc mRNA expression is one of the primary effects of 1,<em>2</em>5(OH)<em>2</em>D3 in the <em>growth</em> inhibition of human <em>keratinocytes</em>.

Fibroblast <em>growth</em> <em>factor</em>-10 (FGF-10) is essential for epithelial development, while other members of this family, such as FGF-7, are not. FGF-10 is abundantly released into wounds following injury, and likely an essential <em>growth</em> <em>factor</em> required for this process. To evaluate how activation of this <em>growth</em> <em>factor</em> is controlled, multiple glycosaminoglycans were combined with FGF-10 assayed by measurement of the proliferation of cell lines expressing FGF receptor-<em>2</em>-IIIb, or <em>keratinocyte</em> migration in an in vitro wound repair assay. Dermatan sulfate (DS) exhibited greater potency than heparan sulfate or other chondroitin sulfates found in wounds. Structural variants of DS between 10 and <em>2</em>0 disaccharides containing iduronic acid showed maximal capacity to enable FGF-10 receptor stimulation. Furthermore, FGF-10 and DS markedly enhanced migration of <em>keratinocytes</em> in an in vitro wound scratch assay, while FGF-7 or other glycosaminoglycans did not. These data strongly suggest that FGF-10 activity is uniquely important in wound repair and that specific DS structural properties are necessary to promote FGF-10 function. These observations identify a novel interplay between DS and FGF-10 in mediating wound repair.

OBJECTIVE

Heat shock protein 90 (Hsp90) is a conserved chaperone involved in crucial signaling events in normal and malignant cells. Previous research suggests that tumor cells are particularly dependent on Hsp90 for survival as well as malignant progression. Hsp90 inhibitors which are derivates of the natural compound geldanamycin, such as the orally bioavailable 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), are currently being tested in clinical trials and small molecule inhibitors are in development. In this study we investigated the response of a panel of cervical carcinoma cell lines in vitro and in vivo to determine potential factors that might influence the sensitivity towards Hsp90 inhibition.

METHODS

Cell viability, proliferation and drug-induced changes on Hsp90 chaperoned "client" factors were examined with focus on G2/M cell cycle regulators, and a comparison with immortalized and normal keratinocytes was performed. ME180 and CaSki cells were grown as subcutaneous xenografts in mice treated with 6-10 mg/kg 17-DMAG by oral gavage 2x/day on a chronic schedule. Tissue concentrations of 17-DMAG were measured by high performance liquid chromatography.

RESULTS

Cell death during abnormal mitosis was observed within 48 h after treatment start. ME180 and CaSki showed more cell death at this time point than SiHa and HeLa, and higher levels of pre-treatment Akt activity. IC(50) values ranged between 17 and 37 nanoM geldanamycin (MTS). Keratinocytes were at least as sensitive as carcinoma cells. All cell lines responded with an increase of the G2/M fraction. Despite in vitro effectiveness and tissue concentrations of 1 microM, only a limited tumor growth reduction was observed with 17-DMAG given close to the maximum tolerated dose level. Lower levels of Hsp90 protein, a lower Akt activity and signs of tissue hypoxia were observed in xenografts compared to cell cultures.

CONCLUSIONS

We show here that Hsp90 inhibition effectively induces apoptosis and growth arrest in cervical carcinoma cells in vitro. Mitotic catastrophe was identified as one mechanism of cell death. In contrast, a limited efficacy of 17-DMAG was observed in subcutaneous xenograft models. Induction of a heat shock response has previously been implicated in resistance towards Hsp90 inhibition. Additional factors might be (1) an altered abundance and/or activity of primary (Hsp90) and secondary (e.g., Akt) target(s), (2) a narrow therapeutic range of 17-DMAG by oral application and (3) response-modifying factors within the tumor environment. The further development of synthetic Hsp90 inhibitors with increased therapeutic window is warranted.

OBJECTIVE

This study aimed to analyze the localization and distribution of vessels and of these angiogenic <em>growth</em> <em>factors</em>: basic fibroblast <em>growth</em> <em>factor</em> (FGF-<em>2</em>), transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha), transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in middle ear cholesteatoma in comparison with normal middle ear mucosa and auditory meatal skin.

BACKGROUND

Angiogenesis is particularly important in many normal and pathologic processes, including wound healing and inflammation. Because proliferating tissues require an enhanced blood supply, angiogenesis appears to be a prerequisite for the expansion of cholesteatoma.

METHODS

The expression of FGF-<em>2</em>, TGF-alpha, TGF-beta1, and VEGF was studied by immunohistochemistry. The amount of vessels (collagen type IV staining) was determined by an automatic imaging analyzing system.

RESULTS

The results showed an altered expression and distribution of VEGF, FGF-<em>2</em>, TGF-alpha, and TGF-beta1 in cholesteatoma in relation to middle ear mucosa and auditory meatal skin. The results were consistent with rapidly growing, activated keratinocytes and stromal cells. Vascularization within the perimatrix of cholesteatoma showed a 4.3-fold increase compared with middle ear mucosa and a twofold increase compared with ear canal skin. An increase of 3.<em>2</em>- to 4-fold in the number of vessels was observed. A close relationship was seen between the density of capillaries, degree of inflammation, and expression of the angiogenic <em>factors</em> investigated, and an increased number of microvessels in cholesteatoma tissue.

CONCLUSIONS

Angiogenesis enables and supports the sustained migration of keratinocytes into the middle ear cavity. Therefore, it is a pivotal factor in the destructive behavior of middle ear cholesteatoma.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a mitogen for rat type II cells and also stimulates differentiation in vitro. Administration of KGF also protects the lung from a variety of injuries and subsequent development of fibrosis. Because transforming <em>growth</em> <em>factor</em> (TGF)-beta has been shown to inhibit epithelial cell proliferation and surfactant protein gene expression in other systems and is thought to be a major effector in pulmonary fibrosis, we sought to determine if TGF-beta would antagonize the effects of KGF in primary cultures of alveolar type II cells. Type II cells were cultured on a matrix of type I collagen and Matrigel in the presence or absence of KGF and/or TGF-beta. KGF alone greatly stimulated proliferation and increased cyclin-dependent kinase (cdk) <em>2</em> kinase activity and Retinoblastoma susceptibility gene product (Rb) phosphorylation. Cyclin D1, cdk<em>2</em>, and cdc<em>2</em>5A protein levels were increased, and p15(Ink4b) and p<em>2</em>7(Kip1) protein levels were decreased. TGF-beta markedly inhibited alveolar epithelial cell proliferation induced by KGF. TGF-beta inhibited cdk<em>2</em> enzyme activity and Rb phosphorylation and increased p15(Ink4b) protein levels. TGF-beta also inhibited differentiation induced by KGF as measured by secretion of surfactant protein-A into the apical media. In summary, TGF-beta inhibits the proliferative effect of KGF in vitro and may be a biologic antagonist of KGF.

Distal lung diseases, such as pulmonary fibrosis or acute lung injury, are commonly associated with local alveolar hypoxia that may be deleterious through the stimulation of alveolar epithelial cell (AEC) apoptosis. In various murine models of alveolar injury, administration of allogenic human mesenchymal stem cells (hMSCs) exerts an overall protective paracrine effect, limiting lung inflammation and fibrosis. However, the precise mechanisms on lung cells themselves remain poorly understood. Here, we investigated whether hMSC-conditioned medium (hMSC-CM) would protect AECs from hypoxia-induced apoptosis and explored the mechanisms involved in this cytoprotective effect. Exposure of rat primary AECs to hypoxia (1.5% O<em>2</em> for <em>2</em>4 h) resulted in hypoxia-inducible <em>factor</em> (HIF)-1α protein stabilization, partly dependent on reactive oxygen species (ROS) accumulation, and in a twofold increase in AEC apoptosis that was prevented by the HIF inhibitor 3-(5'-hydroxymethyl-<em>2</em>'-furyl)-1-benzyl-indazole and the antioxidant drug N-acetyl cysteine. Incubation of AECs with hMSC-CM significantly reduced hypoxia-induced apoptosis. hMSC-CM decreased HIF-1α protein expression, as well as ROS accumulation through an increase in antioxidant enzyme activities. Expression of Bnip3 and CHOP, two proapoptotic targets of HIF-1α and ROS pathways, respectively, was suppressed by hMSC-CM, while Bcl-<em>2</em> expression was restored. The paracrine protective effect of hMSC was partly dependent on <em>keratinocyte</em> <em>growth</em> <em>factor</em> and hepatocyte <em>growth</em> <em>factor</em> secretion, preventing ROS and HIF-1α accumulation.

Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival fibroblasts (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; <em>keratinocyte</em> <em>growth</em> <em>factor</em>-1 (KGF-1), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), and type I collagen production; and oral wound healing in rats. [(3)H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol; and Carbopol containing 0.5%, 1%, and <em>2</em>% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of <em>2</em>, 4, 8, and 16 mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between <em>2</em> - 16 mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of fibroblast proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.