1. We recently demonstrated that ginsenoside Rb1 (C54H9<em>2</em>O<em>2</em>3, molecular weight 1108) isolated from ginseng, when intravenously infused into rats with permanent middle cerebral artery occlusion, reduced cerebral infarct volume and ameliorated place navigation disability of the animals, through an anti-apoptotic action and possibly promotion of vascular regeneration. To investigate the ginsenoside Rb1-mediated vascular regeneration in vivo in a more easily accessible experimental systems, we made a burn wound on the backs of mice and topically applied either Vaseline (vehicle) alone or Vaseline containing low doses of ginsenoside Rb1 to the wound. <em>2</em>. Surprisingly, we found that ginsenoside Rb1 at low concentrations (100 pg g(-1), 1 pg g(-1) and 10 fg g(-1) ointment) exhibited the strongest burn wound-healing action. Furthermore, ginsenoside Rb1 (100 fg-1 ng per wound) increased neovascularization in the surrounding tissue and production of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and interleukin (IL)-1beta from the burn wound, compared to those mice with burn wounds treated with vehicle alone. 3. In human <em>keratinocyte</em> cultures (HaCaT cells), ginsenoside Rb1 (100 fg ml(-1) to 1 ng ml(-1)) enhanced VEGF production induced by IL-1beta and expression of hypoxia-inducible <em>factor</em> (HIF)-1alpha. 4. These findings suggest that the promotion of burn wound healing by ginsenoside Rb1 might be due to the promotion of angiogenesis during skin wound repair via the stimulation of VEGF production, through the increase of HIF-1alpha expression in <em>keratinocytes</em>, and due to the elevation of IL-1beta resulting from the macrophage accumulation in the burn wound.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell <em>growth</em>, migration, and production of MMP-<em>2</em> and -9 in oral mucosal and skin <em>keratinocytes</em> cultured in the presence of synthetic MMP inhibitors. MMP-<em>2</em> was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in <em>keratinocytes</em> stimulated by tumor necrosis <em>factor</em> alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-<em>2</em> but not MMP-9 were able to drastically inhibit migration of both mucosal and skin <em>keratinocytes</em>. Tetracycline analogues also inhibited <em>keratinocyte</em> <em>growth</em>, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-<em>2</em> production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell <em>growth</em> but did inhibit <em>keratinocyte</em> migration. Tumor <em>growth</em> <em>factor</em> beta (TGFbeta) increased <em>keratinocyte</em> migration as well as both cell-associated and secreted MMP-<em>2</em> production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted <em>keratinocyte</em> migration. Immunostaining of <em>keratinocytes</em> advancing into the wound revealed that MMP-<em>2</em> was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-<em>2</em> is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-<em>2</em> plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.

We have reported that normal human salivary gland-derived epithelial cells exclusively express <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR). In the process of malignant transformation of human salivary gland tumors, KGFR gene expression disappeared concomitantly with the de novo expression of the fibroblast <em>growth</em> <em>factor</em> receptor 1 (FGFR1) and FGFR4 genes. In the present study, we introduced wild-type KGFR cDNA or chimeric KGFR/FGFR1 cDNA, which encoded the extracellular domain of KGFR and the intracellular domain of FGFR1, into the HSY human salivary adenocarcinoma cell line. The KGFR tyrosine kinase suppressed the activity of FGF receptor substrate <em>2</em> (FRS<em>2</em>) and inhibited the <em>growth</em> of HSY by inducing differentiation and apoptosis in vitro and in vivo. Our results provided significant insight into the mechanism of KGFR tumor suppression and suggest that KGFR gene therapy might be a viable method of inhibiting human salivary adenocarcinoma <em>growth</em>.

OBJECTIVE

The urinary proteome is a potential easily accessible source of biomarkers for inflammatory bladder diseases, including interstitial cystitis. In the present study, we subjected rat urine to multiplex cytokine analysis in an attempt to identify an inflammatory signature of the temporal course of cyclophosphamide (CYP)-induced cystitis.

METHODS

Rat urine was collected for 1<em>2</em> hours after CYP injection (150 mg/kg) for multiplex analysis of 14 cytokines by a multiple antigen bead assay (Luminex 100 IS). Urine from each void was collected, and the voiding frequency was determined. The bladder tissue was analyzed for cytokines levels and histologic evidence of inflammation.

RESULTS

Significant changes were noted in the urine levels of all cytokines with respect to baseline at <em>2</em>, 4, 6, and 10 hours after CYP injection. Elevation was noted at all times for most cytokines, except for monocyte chemotactic protein-1, which had a 5-fold decrease at <em>2</em> hours. The urine and tissue levels of interleukin (IL)-1beta, IL-4, and <em>growth</em>-related oncogene/<em>keratinocyte</em>-derived chemokine correlated significantly, with a positive Spearman correlation also noted for granulocyte-macrophage colony-stimulating <em>factor</em>, monocyte chemotactic protein-1-1, IL-18, and interferon-gamma. The tissue levels for most cytokines, except for IL-<em>2</em>, and urinary frequency were significantly elevated in the CYP-treated rats compared with the control vehicle-treated rats. The hints of severe inflammation in the bladder indicated by the urinary cytokines were confirmed by bladder histologic examination and the tissue cytokine levels at necropsy.

CONCLUSIONS

The progression of CYP-induced cystitis was clearly reflected in the urine matrix by the temporal and quantitative changes in the cytokine levels. Additional delineation of urine and bladder tissue cytokine expression might yield biomarkers for cystitis.

Psoriasis is an inflammatory skin disease driven by aberrant interactions between the epithelium and the immune system. Anti-psoriatic drugs can therefore target either the <em>keratinocytes</em> or the immunocytes. Here we sought to develop an in vitro reconstructed skin model that would display the molecular characteristics of psoriatic epidermis in a controlled manner, allowing the screening of anti-psoriatic drugs and providing a model in which to study the biology of this disease. Human skin equivalents generated from normal human adult <em>keratinocytes</em> after air exposure and stimulation by <em>keratinocyte</em> <em>growth</em> <em>factor</em> and epidermal <em>growth</em> <em>factor</em> displayed the correct morphological and molecular characteristics of normal human epidermis whereas the psoriasis-associated proteins, hBD-<em>2</em>, SKALP/elafin, and CK16, were absent. Skin equivalents generated from foreskin <em>keratinocytes</em> were clearly abnormal both morphologically and with respect to gene expression. When normal skin equivalents derived from adult <em>keratinocytes</em> were stimulated with psoriasis-associated cytokines [tumor necrosis <em>factor</em>-alpha, interleukin (IL)-1alpha, IL-6, and IL-<em>2</em><em>2</em>] or combinations thereof, strong expression of hBD-<em>2</em>, SKALP/elafin, CK16, IL-8, and tumor necrosis <em>factor</em>-alpha was induced as shown by quantitative polymerase chain reaction and immunohistochemistry. Retinoic acid but not cyclosporin A was found to inhibit cytokine-induced gene expression at both the mRNA and protein levels. These results illustrate the potential of this disease model to study the molecular pathology and pharmacological intervention in vitro.

The kinetics of gene expression associated with the development of cutaneous graft-versus-host disease (GVHD) were examined in a mouse model of MHC-matched allogeneic hematopoietic stem cell transplantation. Ear skin was obtained from recipient mice with or without GVHD between 7 and 40 days after transplantation for histopathological analysis and gene expression profiling. Gene expression patterns were consistent with early infiltration and activation of CD8(+) T and mast cells, followed by CD4(+) T, natural killer, and myeloid cells. The sequential infiltration and activation of effector cells correlated with the histopathological development of cutaneous GVHD and was accompanied by up-regulated expression of many chemokines and their receptors (CXCL-1, -<em>2</em>, -9, and -10; CCL-<em>2</em>, -5, -6, -7, -8, -9, -11, and -19; CCR-1 and CCR-5), adhesion molecules (ICAM-1, CD18, Ly69, PSGL-1, VCAM-1), molecules involved in antigen processing and presentation (TAP1 and TAP<em>2</em>, MHC class I and II, CD80), regulators of apoptosis (granzyme B, caspase 7, Bak1, Bax, and BclII), interferon-inducible genes (STAT1, IRF-1, IIGP, GTPI, IGTP, Ifi<em>2</em>0<em>2</em>A), stimulators of fibroblast proliferation and matrix synthesis (interleukin-1beta, transforming <em>growth</em> <em>factor</em>-beta1), and markers of <em>keratinocyte</em> proliferation (keratins 5 and 6), and differentiation (small proline-rich proteins <em>2</em>E and 1B). Many acute-phase proteins were up-regulated early in murine cutaneous GVHD including serum amyloid A<em>2</em> (SAA<em>2</em>), SAA3, serpins a3g and a3n, secretory leukocyte protease inhibitor, and metallothioneins 1 and <em>2</em>. The kinetics of gene expression were consistent with the evolution of cutaneous pathology as well as with current models of disease progression during cutaneous GVHD.

Directed cell migration is mediated by cycles of protrusion, adhesion, traction generation on the extracellular matrix and retraction. However, how the events after protrusion are timed, and what dictates their temporal order is completely unknown. We used acute epidermal <em>growth</em> <em>factor</em> (EGF) stimulation of epidermal <em>keratinocytes</em> to initiate the cell migration cycle to study the mechanism of the timing of adhesion, traction generation, and de-adhesion. Using microscopic and biochemical assays, we surprisingly found that at approximately <em>2</em> min after EGF stimulation protrusion, activation of myosin-II, traction generation, adhesion assembly, and paxillin phosphorylation occurred nearly simultaneously, followed by a 10-min delay during which paxillin became dephosphorylated before cell retraction. Inhibition of myosin-II blocked both the EGF-stimulated paxillin phosphorylation and cell retraction, and a paxillin phosphomimic blocked retraction. These results suggest that EGF-mediated activation of myosin-II acts as a mechanical signal to promote a cycle of paxillin phosphorylation/dephosphorylation that mediates a cycle of adhesion strengthening and weakening that delays cell retraction. Thus, we reveal for the first time a mechanism by which cells may temporally segregate protrusion, adhesion, and traction generation from retraction during EGF-stimulated cell migration.

Notch signaling plays a key role in cell-fate determination and differentiation in different organisms and cell types. Several reports suggest that Notch signaling may be involved in neoplastic transformation. However, in primary <em>keratinocytes</em>, Notch1 can function as a tumor suppressor. Similarly, in HPV-positive cervical cancer cells, constitutively active Notch1 signaling was found to cause <em>growth</em> suppression. Activated Notch1 in these cells represses viral E6/E7 expression through AP-1 down-modulation, resulting in increased p53 expression and a block of pRb hyperphosphorylation. Here we show that in cervical cancer cell lines in which Notch1 ability to repress AP-1 activity is impaired, Notch1-enforced expression elicits an alternative pathway leading to <em>growth</em> arrest. Indeed, activated Notch1 signaling suppresses activity of the helix-loop-helix transcription <em>factor</em> E47, via ERK1/<em>2</em> activation, resulting in inhibition of cell cycle progression. Moreover, we found that RBP-Jkappa-dependent Notch signaling is specifically repressed in cervical cancer cells and this repression could provide one such mechanism that needs to be activated for cervical carcinogenesis. Finally, we show that inhibition of endogenous Notch1 signaling, although results in a proliferative advantage, sensitizes cervical cancer cell lines to drug-induced apoptosis. Together, our results provide novel molecular insights into Notch1-dependent <em>growth</em> inhibitory effects, counteracting the transforming potential of HPV.

Nerve <em>growth</em> <em>factor</em> (NGF) influences the key pathological events of psoriasis: <em>keratinocyte</em> proliferation, angiogenesis, and T-cell activation. We have systematically examined the kinetics of NGF expression, <em>keratinocyte</em> proliferation, and migration of T lymphocytes in the epidermis in Koebner-induced developing psoriatic plaques. In skin traumatized by the tape-stripping method (n = 1<em>2</em>), a marked up-regulation of NGF in Koebner-positive lesions (n = 7) was observed <em>2</em>4 hours after trauma. Synthesis of NGF reached its maximum level in the <em>2</em>nd week. Furthermore, cultured <em>keratinocytes</em> from nonlesional skin of psoriasis patients produced 10 times higher levels of NGF compared with <em>keratinocytes</em> from healthy individuals. To substantiate the in vivo effect of NGF secreted by <em>keratinocytes</em> in psoriatic plaques, we studied psoriatic plaques and normal human skin in a SCID-human skin xenograft model. The transplanted psoriatic plaques demonstrated marked proliferation of NGF-R (p75)-positive nerve fibers compared with only a few nerves in the transplanted normal human skin. Our results demonstrate that 1) in a developing psoriatic lesion, up-regulation of NGF together with <em>keratinocyte</em> proliferation are early events and precede epidermotropism of T lymphocytes; <em>2</em>) <em>keratinocytes</em> in patients with psoriasis are primed to produce elevated levels of NGF; and 3) NGF synthesized by these <em>keratinocytes</em> is functionally active.

Protein kinase Calpha (PKCalpha) is one of six PKC isoforms expressed in <em>keratinocytes</em> of mouse epidermis. To gain an understanding of the role of epidermal PKCalpha, we have localized its expression to specific cells of normal mouse skin and examined the effect of keratin 5 (K5) promoter directed expression of PKCalpha in transgenic mice. In normal mouse skin, PKCalpha was extensively expressed in the outer root sheath (ORS) <em>keratinocytes</em> of the anagen hair follicle and weakly expressed in <em>keratinocytes</em> of interfollicular epidermis. K5-targeted expression of PKCalpha to epidermal basal <em>keratinocytes</em> and follicular ORS <em>keratinocytes</em> resulted in a tenfold increase in epidermal PKCalpha. K5-PKCalpha mice exhibited no abnormalities in <em>keratinocyte</em> <em>growth</em> and differentiation in the epidermis. However, a single topical treatment with the PKC activator, 1<em>2</em>-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a striking inflammatory response characterized by edema and extensive epidermal infiltration of neutrophils that formed intraepidermal microabscesses in the epidermis. Compared to TPA-treated wild-type mice, the epidermis of TPA-treated K5-PKCalpha mice displayed increased expression of cyclooxygenase-<em>2</em> (COX-<em>2</em>), the neutrophil chemotactic <em>factor</em> macrophage inflammatory protein-<em>2</em> (MIP-<em>2</em>) mRNA and the proinflammatory cytokine TNFalpha mRNA but not IL-6 or IL-1alpha mRNA. To determine if K5-PKCalpha mice display an altered response to TPA-promotion, 7, 1<em>2</em>-dimethylbenz[a]anthracene-initiated K5-PKCalpha mice and wild-type mice were promoted with TPA. No differences in papilloma incidence or multiplicity were observed between K5-PKCalpha mice and wild-type littermates. These results demonstrate that the overexpression of PKCalpha in epidermis increases the expression of specific proinflammatory mediators and induces cutaneous inflammation but has little to no effect on epidermal differentiation, proliferation or TPA tumor promotion.

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT<em>2</em>) cells. We have recently shown that 7-day-old cultures of AT<em>2</em> cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca(<em>2</em>+) changes via gap junctions (<em>2</em>5). In this study, we cultured AT<em>2</em> cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of <em>keratinocyte</em> <em>growth</em> <em>factor</em>. Under these conditions, cultured AT<em>2</em> cells display AT<em>2</em> type morphology, express the AT<em>2</em>-specific marker surfactant protein C, and do not express AT1-specific cell marker aquaporin 5, all consistent with maintenance of AT<em>2</em> phenotype. These AT<em>2</em>-like cells also coordinate mechanically induced intercellular Ca(<em>2</em>+) signaling, but, unlike AT1-like cells, do so by using extracellular nucleotide triphosphate release. Additionally, cultured cells that retain AT<em>2</em> cell-specific markers express connexin profiles different from cultured cells with AT1 characteristics. The parallel changes in intercellular Ca(<em>2</em>+) signaling with cell differentiation suggest that cell signaling mechanisms are an intrinsic component of lung alveolar cell phenotype. Because lung epithelial injury is accompanied by extracellular matrix and <em>growth</em> <em>factor</em> changes, followed by extensive cell division, differentiation, and migration of AT<em>2</em> progenitor cells, we suggest that similar changes may be vital to the lung recovery and repair process in vivo.

Lung branching morphogenesis depends on mesenchymal-epithelial tissue interactions. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has been implicated to be a regulator of these tissue interactions. In the present study, we investigated the role of KGF in early rat lung organogenesis. Reverse transcriptase-polymerase chain reaction analysis revealed KGF mRNA expression in the mesenchymal component of the 13-day embryonic lung, while message for KGF receptor (KGFR) was expressed in the epithelium, confirming the paracrine nature of KGF/KGFR axis. Antisense KGF oligonucleotides inhibited DNA synthesis of embryonic lung explants. This inhibitory effect of antisense KGF was partially reversed by the addition of exogenous KGF. Recombinant KGF was mitogenic for 13-day isolated embryonic lung epithelial cells. Medium conditioned by 13-day lung mesenchymal cells also stimulated DNA synthesis of 13-day embryonic lung epithelial cells. This stimulatory effect was partially abrogated by a neutralizing KGF antibody. The number of terminal buds of lung explants cultured in the presence of antisense KGF oligonucleotides was significantly reduced compared to control explants. Exogenous KGF partially abrogated the inhibitory effect of antisense KGF on early lung branching. Sense or scrambled KGF oligonucleotides had no inhibitory effect on lung <em>growth</em> and branching. Addition of neutralizing KGF antibodies to the explants also reduced the degree of branching, while non-immune IgG and neutralizing acidic FGF antibodies had no effect. Explants incubated with antisense oligonucleotides targeted to the initiation site of translation of both the splice variants of the fibroblast <em>growth</em> <em>factor</em> receptor-<em>2</em> (FGFR<em>2</em>) gene, KGFR and bek, exhibited a similar reduction in lung branching as observed with antisense KGF oligonucleotides. Antisense KGFR-specific oligonucleotides dramatically inhibited lung branching, while exposure of explants to antisense bek-specific oligonucleotides resulted in reduced branching albeit to a lesser degree than that observed with antisense KGFR-specific oligonucleotides. Neither sense nor scrambled KGFR-specific oligonucleotides had any effect on early lung branching. These results suggest that the KGF/KGFR system has a critical role in early lung organogenesis.

OBJECTIVE

The use of nuclear/radiation devices against the civilian population is now a realistic scenario. Haematopoietic syndrome is the primary therapeutic challenge in the case of whole body acute exposure over <em>2</em> Grays (Gy) whereas burns and combined injuries would be frequently observed in myelo-suppressed patients. Optimisation of scoring and treatments are important goals to achieve.

CONCLUSIONS

The European Response Category (RC) concept represents an attempt to integratively assess haematological/extrahematological radiation-induced lesions. Based on the frequently observed heterogeneity of bone marrow damage in accidental/intentional irradiations, the stimulation of residual stem cells using granulocyte Colony-stimulating factor remains the therapeutic standard after exposure to less than the lethal dose 50 % (Haematopoietic[H] score 3-H3). Allogeneic stem cell transplantation is indicated in case of medullary eradication (Haematopoietic score 4-H4) whereas extramedullary toxicity may determine the outcome. Especially in case of numerous casualties exhibiting acute radiation syndrome, the administration of survival factor combinations remains questionable, at least as a palliative treatment. In addition pleiotropic cytokines injection such as erythropoietin and keratinocyte growth factor and grafting multipotent mesenchymal stem cells - from underexposed bone marrow areas or fat tissues - could be proposed to prevent multiple organ failure syndrome development. Multi-disciplinary teams should be prepared to manage such patients.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) is known to stimulate dermal wound healing events (fibroplasia and fibrosis). In this study, the effect of TGF-beta on epidermal wound healing (re-epithelialization) was examined. Epidermal cell out<em>growth</em> from partial-thickness porcine skin explants was used as an in vitro model for epithelialization. All cultures were grown in medium with 1% fetal bovine serum, which was sufficient for explant viability but low enough to permit measurement of modulation by added <em>factors</em>. Because TGF-beta is known to act in concert with other <em>growth</em> <em>factors</em>, it was evaluated alone and in the presence of epidermal <em>growth</em> <em>factor</em> (EGF) and platelet-derived <em>growth</em> <em>factor</em> (PDGF). The results indicate that TGF-beta produced earlier initiation of out<em>growth</em>, by 1-<em>2</em> d compared with control cultures, and increased the rate of out<em>growth</em> during the migratory phase of culture (Days 1-3). Compared to controls, EGF alone produced a greater percentage of <em>growing</em> explants and an increased rate of out<em>growth</em> during the mitotic phase (Days 4-7). TGF-beta (1 or 10 ng/ml) and EGF (5 ng/ml) had an additive rather than a synergistic effect on out<em>growth</em>. PDGF-treated explants did not show enhanced <em>growth</em> when PDGF (<em>2</em>.5 units/ml) was added alone or together with TGF-beta and EGF. The ability of TGF-beta to produce earlier initiation of out<em>growth</em> was not due to an effect on mitosis, because TGF-beta did not increase the incorporation of [3H]thymidine into <em>keratinocytes</em> in the <em>growing</em> epidermal sheets. Rather, it is likely that TGF-beta facilitated <em>keratinocyte</em> migration, possibly by unmasking a receptor on the epidermal cell surface. These results suggest that TGF-beta may play a role in early epidermal wound healing.

Epidermal <em>keratinocytes</em> are continuously exposed to mechanical forces. The human skin surface can be thickened and enlarged by various stresses such as tissue expander or abrasive pressure. To investigate the mechanism of epidermal hyperproliferation by mechanical stress, <em>keratinocytes</em> were plated on flexible silicone dishes, which were continuously stretched by +<em>2</em>0%. Stretching of cells for <em>2</em>4 h caused upregulation of 5-bromo-<em>2</em>'-deoxyuridine (BrdU)-positive cells to <em>2</em>00%-<em>2</em><em>2</em>0% and activation of extracellular signal-regulated kinases (ERK)1/<em>2</em>. Inhibition of mitogen and ERK with U01<em>2</em>6 and phosphoinositide 3-OH kinase attenuated BrdU incorporation and ERK1/<em>2</em> activation. The EGF receptor kinase inhibitor and the calcium channel inhibitor also inhibited BrdU incorporation and the activation of ERK1/<em>2</em>. Twenty-four hours of stretching stimulated reporter activity driven by activator protein 1 (AP-1), induction of K6, and suppression of K10, which were inhibited by U01<em>2</em>6. Our results indicate that mechanical stretching induces proliferative signals on human <em>keratinocytes</em> via induction of calcium influx, phosphorylation of epidermal <em>growth</em> <em>factor</em> receptor (EGFR), and ERK1/<em>2</em>. These mechanisms may contribute to the hyperproliferative nature of the epidermis, which is mechanically stretched by various stimuli.

Cultures of differentiated melanocytes can readily be grown from the dissociated epidermis of neonatal mice, and immortal cell lines often develop from these. However, the first cells that grow and transiently dominate the cultures, while similar to melanocytes, are unpigmented. These have been shown to be precursors of melanocytes and may be termed melanoblasts. Under our previous standard culture conditions, involving the use of <em>keratinocyte</em> feeder cells, foetal calf serum, the phorbol ester 1<em>2</em>-O-tetradecanoyl phorbol acetate (TPA) and cholera toxin, all the melanoblasts spontaneously differentiated to pigmented melanocytes within about 3 weeks. We now describe some <em>factors</em> affecting the proliferation and differentiation of diploid murine melanoblasts in the presence of serum. Murine stem cell <em>factor</em>/steel <em>factor</em> (SCF), basic fibroblast <em>growth</em> <em>factor</em> (bFGF) and murine leukaemia inhibitory <em>factor</em>/differentiation-inhibiting activity (LIF/DIA) all increased melanoblast numbers. SCF and LIF also slightly inhibited melanoblast differentiation, while cholera toxin and TPA promoted differentiation. Using some of these findings, and by regular replacement of <em>keratinocyte</em> or fibroblastoid feeder cells, we have established a clonal line of immortal murine melanoblasts, 'melb-a'. These cells express tyrosinase-related protein-<em>2</em> but not, in general, tyrosinase. They can be induced to differentiate irreversibly to functional melanocytes (also proliferative and immortal) by plating in the absence of feeder cells. Thus a new immortal melanocyte line, 'melan-a<em>2</em>', has also been produced.

The androgen receptor (AR) is expressed in differentiated secretory prostate epithelial cells in vivo. However, in the human prostate, it is unclear whether androgens directly promote the survival of secretory cells, or whether secretory cells survive through androgen-dependent signals from the prostate stroma. Biochemical and mechanistic studies have been hampered by inadequate cell-culture models. In particular, large-scale differentiation of prostate epithelial cells in culture has been difficult to achieve. Here, we describe the development of a differentiation system that is amenable to functional and biochemical analysis and its application to deciphering the survival pathways in differentiated AR-expressing epithelial cells. Confluent prostate epithelial cell cultures were treated with <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and dihydrotestosterone. After <em>2</em> weeks, a suprabasal cell layer was formed in which cells no longer expressed alpha<em>2</em>, alpha3, alpha6, alphav, beta1 or beta4 integrins or p63, K5, K14, EGFR, FGFR<em>2</em>IIIb or Bcl-<em>2</em>, but instead expressed AR and androgen-induced differentiation markers, including K18, K19, TMPRSS<em>2</em>, Nkx3.1, PMSA, KLK<em>2</em> and secreted prostate-specific antigen (PSA). Differentiated prostate cell survival depended on E-cadherin and PI3K, but not KGF, androgen, AR or MAPK. Thus survival of differentiated prostate epithelial cells is mediated by cell-cell adhesion, and not through androgen activity or prostate stroma-derived KGF.

Gene amplification and protein overexpression of fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>) characterize the SUM-5<em>2</em> breast cancer cell line developed in our laboratory. SUM-5<em>2</em> cells express nine distinct alternatively spliced isoforms of FGFR<em>2</em>. Among these isoforms are two otherwise identical FGFR<em>2</em> variants that express either the C1 or C3 carboxyl terminus. FGFR<em>2</em>-C3 variants are not normally expressed by human mammary epithelial (HME) cells, and we have shown that overexpression of FGFR<em>2</em>-C3 in HME cells results in potent transformation. In particular, FGFR<em>2</em>-C3 expression leads to robust levels of constitutively tyrosine phosphorylated FRS<em>2</em> in the absence of ligand stimulation. In contrast, overexpressed FGFR<em>2</em>-C1 requires constant stimulation with exogenous <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) to mimic the signaling capability of FGFR<em>2</em>-C3. However, activation of FRS<em>2</em> that results from KGF-stimulated FGFR<em>2</em>-C1 signaling is transient and is associated with a mobility shift of FRS<em>2</em> not observed when this signaling molecule is activated by the C3 isoform of FGFR<em>2</em>. Mutation of the only tyrosine phosphorylated site present in the C1 terminus and absent from C3, Tyr769, did not yield a receptor that rivaled the potent signaling of FGFR<em>2</em>-C3. We therefore conclude that aberrant expression of alternatively spliced isoforms of FGFR<em>2</em> with the C3 carboxyl terminus in the SUM-5<em>2</em> breast cancer cells results in sustained activation of signal transduction leading to transformation.

OBJECTIVE

Acute mucositis is a dose-limiting toxicity of concurrent chemoradiotherapy regimens for locally advanced head and neck cancer. Palifermin (a recombinant human keratinocyte growth factor; DeltaN23-KGF) stimulates the proliferation and differentiation of mucosal epithelium to reduce mucositis in patients receiving intensive therapy for hematologic cancers. This study assessed the efficacy and safety of palifermin in patients receiving concurrent chemoradiotherapy for advanced head and neck squamous cell carcinoma.

METHODS

In a phase II trial, standard radiotherapy was delivered in daily 2-Gy fractions to 70 Gy, or hyperfractionated radiotherapy was delivered in 1.25-Gy fractions twice daily to 72 Gy, over 7 weeks. Chemotherapy included cisplatin 20 mg/m(2) for 4 days and continuous-infusion fluorouracil 1,000 mg/m(2)/d for 4 days on weeks 1 and 5 of irradiation. Patients were randomly assigned 2:1 to palifermin 60 microg/kg or placebo once weekly for 10 doses. A follow-up trial evaluated long-term survival.

RESULTS

Sixty-seven patients received palifermin and 32 received placebo. The median duration of grade>>or= 2 mucositis was 6.5 and 8.1 weeks in the palifermin and placebo groups, respectively (P = .157). Palifermin appeared to reduce mucositis, dysphagia, and xerostomia during hyperfractionated radiotherapy (n = 40) but not standard radiation therapy (n = 59). Adverse events were similar between treatment groups. Palifermin did not alter tumor response or survival.

CONCLUSIONS

Ten once-weekly doses of palifermin at 60 microg/kg were well tolerated. Most patients completed treatment, but palifermin did not reduce the morbidity of concurrent chemotherapy and radiotherapy. Future studies should evaluate higher palifermin doses with longer and more standardized assessment of acute mucositis.

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human <em>keratinocytes</em> were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm<em>2</em> energy doses. Irradiations were done every <em>2</em>4 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm<em>2</em>. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm<em>2</em>. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the <em>keratinocytes</em>. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) gene expression, especially for 1.5 J/cm<em>2</em>, but no change on fibroblast <em>growth</em> <em>factor</em>-<em>2</em> (FGF-<em>2</em>) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm<em>2</em> promoted the most significant biostimulatory effects on cultured <em>keratinocytes</em>.

The human papillomavirus type 16 E5 gene product has been shown to upregulate the activation of MAP kinases ERK1/<em>2</em> and cellular proliferation promoted by EGF in a ligand-dependent process. We now report the <em>growth</em> <em>factor</em>-independent modulation of MAP kinases by HPV 16 E5 in human <em>keratinocytes</em>. After treatment with 600 mM sorbitol or low concentrations of anisomycin, E5-expressing cells upregulate the activation of ERK1/<em>2</em>. In addition, E5 enhances p38 activation after anisomycin but not after sorbitol treatment, but it has no effect on MAP kinases activation after shocking the cells with 300 mM sodium chloride. The E5-mediated, sorbitol-dependent increase in ERK1/<em>2</em> activation is EGF-independent and is only partially inhibited by tyrphostin AG1478, which is known to inhibit specifically EGF receptor activation.

BACKGROUND

Previous studies have demonstrated that nerve growth factor (NGF) is an important mediator of pathologic pain. Many studies have focused on cutaneous mechanisms for NGF-induced hyperalgesia; few have examined its contribution in deeper tissues like muscle. This study examined pain behaviors and the expression of NGF in incised hind paw flexor digitorum brevis muscle.

METHODS

Adult Sprague-Dawley rats were pretreated with anti-NGF peptibody and underwent skin or skin plus deep fascia and muscle incision. Guarding pain behaviors were measured. Muscle NGF messenger RNA (mRNA) was measured by reverse-transcriptase polymerase chain reaction. Changes in NGF protein expression were measured using Western blot, enzyme-linked immunosorbent assay, and immunohistochemistry. In situ hybridization for NGF mRNA was also performed.

RESULTS

Pretreatment with anti-NGF peptibody (100 mg/kg) decreased the guarding behavior caused by deep fascia and muscle incision. Muscle NGF mRNA increased abruptly 2 h after incision and was the same as control by postoperative day 1. NGF protein increased from 4 h after incision and was sustained for several days. NGF was localized in many calcitonin gene-related peptide-positive axons, few N52-positive axons, but not isolectin B4-positive axons in incised muscle. The sources of NGF mRNA included keratinocytes in epidermis and fibroblasts in deeper tissues.

CONCLUSIONS

Fibroblasts adjacent to the injury are sources of NGF in incised muscle. NGF is upregulated by incision of muscle and contributes to guarding pain behavior.

Studies of secretion of surfactant proteins by alveolar type II cells have been limited because the expression of the genes for these proteins decreases rapidly in primary culture. We developed a culture system to investigate the regulation of lipid and protein secretion by alveolar type II cells and the genes involved in these processes. Rat type II cells were plated on membrane inserts coated with rat-tail collagen in medium containing 10% fetal bovine serum (FBS) for 1 d before being changed to medium containing 5 ng/ml <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and <em>2</em>% serum for 3 d and to medium with 5% Engelbreth-Holm-Swarm tumor matrix (EHS) but without serum for <em>2</em> d. From this time forward, the cells were placed on a rocking platform and cultured with 0.4 ml medium on the apical surface at the air-liquid interface (A/L) in four different, serum-free media: basal Dulbecco's modified Eagle's medium (DMEM)/F1<em>2</em> medium (DF1<em>2</em>), basal medium plus EHS (DF1<em>2</em>/EHS), basal medium plus KGF (DF1<em>2</em>/KGF), and basal medium plus EHS and KGF (DF1<em>2</em>/EHS/KGF). Cells cultured in DF1<em>2</em> and DF1<em>2</em>/EHS assumed an attenuated, flattened morphology, whereas those in DF1<em>2</em>/KGF and DF1<em>2</em>/EHS/KGF were more cuboidal, contained numerous lamellar bodies, and had apical microvilli. Cells cultured in DF1<em>2</em> and DF1<em>2</em>/EHS produced a relatively weak signal for the surfactant protein mRNAs (surfactant proteins [SP]-A, SP-B, SP-C, and SP-D, respectively), and secretion of SP-A and SP-D remained low. In contrast, cells maintained for 3 d at A/L and cultured in the presence of KGF showed strong signals for SP-A, SP-B, and SP-D mRNAs, and secreted SP-A, SP-D, and lysozyme into the apical medium. The combination of 1<em>2</em>-O-tetradecanoyl-phorbol-11-acetate (TPA) and terbutaline stimulated secretion of [3H]phosphatidylcholine ([3H]PC), SP-A, and lysozyme, but not SP-D. This primary culture system should prove useful for mechanistic studies of the secretion of SP-A, SP-D, and surfactant lipids.

BACKGROUND

Tumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors.

METHODS

4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted.

RESULTS

The co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45+ leukocytes into tumor tissues. The proportions of Ki-67+ proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone.

CONCLUSIONS

The crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis.