Biomedical subjects

N E Fusenig

Publications and source records attributed to N E Fusenig.

At least 19 recordsLinked to original sources

PubMed2002

Carcinoma-like vascular density in atypic keratoacanthoma suggests malignant progression.

Differential diagnosis between keratoacanthomas and well differentiated squamous cell carcinomas based on clinical and histomorphological data is problematic. Recent findings of cellular atypia in a large proportion of keratoacanthomas indicated that these potentially 'self-healing' cutaneous neoplasms had the potential for malignant progression. Another malignancy-associated criterion is enhanced angiogenesis with increased microvessel density. To provide further diagnostic markers for keratoacanthomas we examined microvessel density on paraffin sections of 13 keratoacanthomas in comparison with 10 normal skin biopsies and 16 late-stage skin squamous cell carcinomas by counting and by computer-assisted image analysis of CD31-immunostained vessels. A significant increase of microvessel density in 'hot spots' was observed in keratoacanthomas as compared to normal skin. Furthermore, when keratoacanthomas were subdivided into tumours with and without malignancy-associated atypic areas, only those with atypia (n=6) were significantly better vascularised than normal skin and had a mean microvessel density in the range of late-stage squamous cell carcinomas. Both keratoacanthoma subtypes revealed comparable levels of inflammatory cell infiltration, tumour cell proliferation and vascular endothelial growth factor expression (mRNA and protein). Thus, in addition to malignancy-associated cellular atypia, increased microvessel density may serve as further diagnostic parameter to discriminate keratoacanthomas with a potential to progress to malignancy.

Biopsy↗

PubMed2002

Magnetic resonance imaging of nude mice with heterotransplanted high-grade squamous cell carcinomas: use of a low-loaded, covalently bound Gd-Hsa conjugate as contrast agent with high tumor affinity.

RATIONALE: Malignant tumors often show an increased uptake and metabolism of plasma proteins, especially albumin. OBJECTIVES: Determine whether the accumulation of low loaded Gd-albumin improves visualization of malignant tumors by MRI. METHODS: Twelve nude mice with heterotransplanted squamous cell carcinomas were studied. The signal intensity of tumor, blood, liver, kidney and muscle tissue was studied in MR images after application of Gd-albumin during a period of 144 hours. MRI results were histologically correlated after simultaneously injection of Gd- and fluorescein-labeled albumins in 9 nude mice. RESULTS: Although liver and kidney had a maximum increase in signal intensity within 30 minutes, tumors showed a delayed 51% increase in the 24 hours after application. Histologic and fluorescence evaluation demonstrated albumin localization in tumors predominantly in stroma and necroses. CONCLUSIONS: Gd-albumin is efficiently accumulated in SCC transplants. MRI with low loaded Gd-albumin may offer relevant opportunities for recognizing tumors sensitive to a therapy with cyostic drug-labeled albumins.

Albumins↗

PubMed2001

The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies.

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.

Angiogenesis Inhibitors↗

PubMed2001

Tumor progression of skin carcinoma cells in vivo promoted by clonal selection, mutagenesis, and autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

Tumor microenvironment is crucial for cancer growth and progression as evidenced by reports on the significance of tumor angiogenesis and stromal cells. Using the HaCaT/HaCaT-ras human skin carcinogenesis model, we studied tumor progression from benign tumors to highly malignant squamous cell carcinomas. Progression of tumorigenic HaCaT-ras clones to more aggressive and eventually metastatic phenotypes was reproducibly achieved by their in vivo growth as subcutaneous tumors in nude mice. Their enhanced malignant phenotype was stably maintained in recultured tumor cells that represented, identified by chromosomal analysis, a distinct subpopulation of the parental line. Additional mutagenic effects were apparent in genetic alterations involving chromosomes 11 and 2, and in amplification and overexpression of the H-ras oncogene. Importantly, in vitro clonal selection of benign and malignant cell lines never resulted in late-stage malignant clones, indicating the importance of the in vivo environment in promoting an enhanced malignant phenotype. Independently of their H-ras status, all in vivo-progressed tumor cell lines (five of five) exhibited a constitutive and stable expression of the hematopoietic growth factors granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which may function as autocrine/paracrine mediators of tumor progression in vivo. Thus, malignant progression favored by the in vivo microenvironment requires both clonal selection of subpopulations adapted to in vivo growth and mutational events leading to stable functional alterations.

Autocrine Communication↗

PubMed2001

Suicide gene therapy for premalignant disease: a new strategy for the treatment of intraepithelial neoplasia.

The potential of gene therapy to treat premalignant disease or recurrent cancer has not been investigated. The goal of the present investigation was to explore the efficacy of pro-drug-mediated, suicide gene therapy as a strategy to treat incipient neoplasia in stratified squamous epithelium. To test this strategy, a tissue model of premalignancy was generated by mixing normal human keratinocytes (NHK) that express the bacterial cytosine deaminase gene (CD) with premalignant keratinocytes which have been genetically marked with the bacterial gene for beta-galactosidase (II-4-beta-gal) in skin-like organotypic cultures. Preliminary studies in monolayer cultures demonstrated that CD-transduced NHK (NHK/CD) efficiently expressed the transgene and deaminated the pro-drug 5-fluorocytosine (5FC) to the toxic product 5-fluorouracil (5FU). The capacity of NHK/CD to kill II-4-beta-gal cells through bystander effect was assayed in both submerged culture and in the organotypic model of premalignancy. In submerged cultures, it was found that CD-mediated killing of II-4-beta-gal cells did not require cell-cell contact and that the LD(50) of 5FC for efficient bystander killing of II-4-beta-gal was 0.5 mM. When this concentration of pro-drug was used in organotypic cultures, a significant number of dysplastic II-4-beta-gal cells were eliminated from the tissue. Bystander killing of II-4-beta-gal cells was related to the number of NHK/CD present. These findings demonstrated that potentially malignant keratinocytes could be eliminated from a dysplastic tissue through activation of pro-drug and killing of adjacent cells through the bystander effect. By establishing an in vitro model to eliminate premalignant cells using suicide gene therapy, these studies provide a new approach for the treatment of incipient cancer as it develops, thereby preventing invasive disease.

Antimetabolites↗

PubMed2001

Differential stromal regulation of MMP-1 expression in benign and malignant keratinocytes.

There is accumulating evidence of the critical role of tumor stroma in carcinoma development and progression. We have studied the significance of stromal components in regulating matrix metalloproteinases in different stages of human skin carcinogenesis using the HaCaT keratinocyte transformation model. Expression of matrix metalloproteinase 1 and matrix metalloproteinase 13 was analyzed in nontumorigenic HaCaT cells and their c-Ha-ras-transformed tumorigenic clones, benign A-5 and malignant A-5RT3, in response to different matrices and cocultured fibroblasts as well as in transplants in nude mice. When cultured on a collagen type I gel, expression of matrix metalloproteinase 1 mRNA was induced in A-5 and A-5RT3 but less in HaCaT cells, whereas matrix metalloproteinase 13 was only induced in A-5 cells. Induction of matrix metalloproteinase 1 by collagen was also observed in two other malignant HaCaT-ras clones as well as in 2/2 primary squamous cell carcinoma lines. In organotypic cocultures with skin fibroblasts, matrix metalloproteinase 1 mRNA and protein was further strongly upregulated in A-5RT3 cells but less in HaCaT and A-5 cells. Importantly, matrix metalloproteinase 1 was also upregulated in fibroblasts when cocultured with A-5RT3 cells. In vivo, A-5RT3 transplants and subcutaneous tumors expressed matrix metalloproteinase 1 mRNA consistently, preferentially at the tumor front to the mouse stroma. In contrast, matrix metalloproteinase 1 expression was absent in the transplants of A-5 cells and HaCaT cells. Thus, our results demonstrate the specific induction of matrix metalloproteinase 1 in malignant keratinocytes by fibroblasts, supposedly through paracrine-acting factors, and a reciprocally enhanced expression in fibroblasts. This further substantiates the important role of tumor stroma in regulating the expression of matrix metalloproteinase 1, a major matrix-degrading proteinase implicated in tumor invasion.

Cell Line, Transformed↗

PubMed2001

Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation.

Organotypic cocultures of keratinocytes and fibroblasts generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The use of mouse fibroblasts and human keratinocytes facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and growth regulation. Moreover, the functional significance for the keratinocyte phenotype of genetically modified fibroblasts from transgenic or knockout mice, even those exhibiting an embryonic lethal phenotype, can be studied in such heterologous in vitro tissue equivalents. Here we communicate results of such studies revealing the antagonistic function of mouse fibroblasts defective in the AP-1 constituents c-Jun and JunB, respectively, on human keratinocyte growth and differentiation. Furthermore, the hematopoietic growth factor granulocyte macrophage-colony stimulating factor has been identified as a novel regulator of keratinocyte growth and differentiation. As will be reported in detail elsewhere both granulocyte macrophage-colony stimulating factor and keratinocyte growth factor have been identified as major mediators of fibroblast-keratinocyte interactions and their expression is induced via AP-1 by interleukin-1 released by the epithelial cells. Thus, these heterologous cocultures provide a novel promising tool for elucidating molecular mechanisms of epithelial-mesenchymal interactions and their consequences on epithelial cell proliferation and differentiation.

Adult↗

PubMed2000

c-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin.

Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, we reconstituted skin by combining primary human keratinocytes and mouse wild-type, c-jun(-/-), and junB(-/-) fibroblasts. We have discovered an antagonistic function of these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation, and traced this effect to the IL-1-dependent regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion directs regeneration of the epidermis and maintenance of tissue homeostasis in skin.

Animals↗

PubMed2000

Inhibition of collagenase-3 (MMP-13) expression in transformed human keratinocytes by interferon-gamma is associated with activation of extracellular signal-regulated kinase-1,2 and STAT1.

Collagenase-3 (MMP-13) is characterized by an exceptionally wide substrate specificity and restricted expression. MMP-13 is specifically expressed by transformed human keratinocytes in squamous cell carcinomas in vivo and its expression correlates with their invasion capacity. Here, we show, that interferon-gamma (IFN-gamma) markedly inhibits expression of MMP-13 by human cutaneous SCC cells (UT-SCC-7) and by ras-transformed human epidermal keratinocytes (A-5 cells) at the transcriptional level. In addition, IFN-gamma inhibits collagenase-1 (MMP-1) expression in these cells. IFN-gamma abolished the enhancement of MMP-13 and MMP-1 expression by transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha), and inhibited invasion of A-5 cells through type I collagen. IFN-gamma also rapidly and transiently activates extracellular signal-regulated kinase 1,2 (ERK1,2) and blocking ERK1,2 pathway (Raf/MEK1,2/ERK1,2) by specific MEK1,2 inhibitor PD98059 partially (by 50%) prevents Ser-727 phosphorylation of STAT1 and suppression of MMP-13 expression by IFN-gamma. Furthermore, Ser-727 phosphorylation of STAT1 by ERK1,2, or independently of ERK1,2 activation is associated with marked reduction in MMP-13 expression. These observations identify a novel role for IFN-gamma as a potent inhibitor of collagenolytic activity and invasion of transformed squamous epithelial cells, and show that inhibition of MMP-13 expression by IFN-gamma involves activation of ERK1,2 and STAT1.

Cell Line, Transformed↗

PubMed2000

TGF-beta isoforms are differentially expressed in increasing malignant grades of HaCaT keratinocytes, suggesting separate roles in skin carcinogenesis.

The three mammalian isoforms of transforming growth factor-beta (TGF-beta1, -beta2, and -beta3) are potent regulators of cell growth, differentiation, and extracellular matrix deposition. To study their role in skin carcinogenesis, normal human keratinocytes, early (31) and late (310) passage immortalized keratinocytes (HaCaT cells), and five HaCaT-ras clones exhibiting benign (A-5, I-7), malignant (II-4, A-5 RT1), and highly aggressive (A-5 RT3) tumourigenic phenotypes were examined for the expression of TGF-beta isoforms, by immunohistochemistry. This was performed under in vivo conditions, in surface transplants and subcutaneously growing tumours in nude mice. Generally, all tissues that formed keratinized epithelia demonstrated an immunostaining pattern similar to normal human skin. TGF-beta1 was localized to the upper differentiated layers, the stratum granulosum and corneum, in a perimembranous pattern, whereas TGF-beta2 and, weaker, TGF-beta3 immunostaining was present in all suprabasal layers of normal keratinizing epithelia. In contrast, non-keratinizing transplants of non-tumourigenic or highly aggressive cells showed little to no immunoreactivity for TGF-beta1. Whereas TGF-beta2 expression was moderate in the upper layers of non-tumourigenic epithelia, large tumour cells of the malignant HaCaT-ras clones, particularly at the invasion front, were strongly positive for TGF-beta2. TGF-beta3 immunostaining was most pronounced in the stroma of malignant tumours, implying its paracrine induction by the malignant tumour transplants. These results suggest differential functions for each TGF-beta isoform in epidermal carcinogenesis, such that TGF-beta1 is associated with the more differentiated state, TGF-beta2 with highly malignant and invading cells, and TGF-beta3 with tumour stroma formation and angiogenesis. Furthermore, the expression of TGF-betas by both early- and late-stage tumours implies that the isoforms may have distinct functions at different stages of malignancy, supporting their dual role in skin carcinogenesis.

Animals↗

PubMed2000

Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced keratinocyte growth factor expression in resting fibroblasts.

Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal-epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitotic (irradiated) fibroblasts were incorporated in the collagen matrix, where they typically support epidermal proliferation and tissue organization. In coculture with keratinocytes, fibroblasts exhibit an enhanced expression of keratinocyte growth factor and the interleukin-1 receptor (type I), which further increase with culture time. In cocultured keratinocytes, keratinocyte growth factor receptor as well as RNA expression and protein release of interleukin-1alpha and interleukin-1beta are upregulated. We hypothesized that the modulated cytokine expression represents a basic mechanism for keratinocyte growth regulation. The functional significance of this double paracrine pathway, i.e., induction of keratinocyte growth factor expression in fibroblasts by keratinocytes via release of interleukin-1, was confirmed by interfering with both signaling elements: (i) interleukin-1-neutralizing antibodies and interleukin-1 receptor antagonist significantly inhibited keratinocyte growth factor release, keratinocyte proliferation, and tissue formation comparable to the effect produced by keratinocyte-growth-factor-blocking antibodies; (ii) addition of keratinocyte growth factor to cocultures with inactivated interleukin-1 pathway completely reverted growth inhibition; (iii) in organotypic cocultures with subthreshold fibroblast numbers both interleukin-1 and keratinocyte growth factor restored the impaired epidermal morphogenesis. Thus, epidermal tissue regeneration in organotypic cocultures is mainly regulated by keratinocyte-derived interleukin-1 signaling, which induces keratinocyte growth factor expression in cocultured fibroblasts. This demonstrates a novel role for interleukin-1 in skin homeostasis substantiating data from wound healing studies in vivo.

Cell Differentiation↗

PubMed2000

Angiogenic switch occurs late in squamous cell carcinomas of human skin.

Angiogenesis is a crucial event in carcinogenesis and its onset has been associated with premalignant tumour stages. In order to elucidate the significance of angiogenesis in different stages of epithelial skin tumours, we analysed the vessel density in ten normal skin samples, 14 actinic keratosis (AK), 12 hypertrophic AKs, and in nine early- and 16 late-stage squamous cell carcinomas (SCCs). Mean vascular density was quantitated by counting the number of CD 31-immunostained blood vessels and by morphometric assessment of stained vessel area by computer-assisted image analysis. The results from both methods were well correlated. Mean vascular density was similar in normal dermis and in AK, and only slightly elevated in hypertrophic AKs and early SCC stages (tumour thickness < 2 mm). Only late-stage SCCs infiltrating the subcutis exhibited a significant increase in vascularization. Vessel density was independent of tumour localization, degree of proliferation and inflammatory cell infiltration. Furthermore, tumour vascularization was not correlated with the expression of vascular endothelial growth factor, a major angiogenic factor, as revealed by in situ hybridization and immunohistochemistry. The restriction of enhanced vascularization to increased tumour thickness may be a major reason for the rather low metastatic spread of cutaneous SCCs.

Carcinoma, Squamous Cell↗

PubMed2000

Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase.

Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by transformed squamous epithelial cells, i.e. squamous cell carcinoma (SCC) cells in culture and in vivo. Here, we have elucidated the signaling pathways regulating MMP-13 expression in transformed human epidermal keratinocytes, i.e. ras-transformed HaCaT cell line A-5 and cutaneous SCC cell line (UT-SCC-7). Treatment with tumor necrosis factor-(alpha) (TNF-(alpha) resulted in activation of extracellular signal-regulated kinase (ERK)1,2, Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) in both cell lines. In addition, transforming growth factor-(beta) (TGF-(beta) activated p38 MAPK in both cell lines, and ERK2 in A-5 cells. Selective inhibition of p38 activity with SB 203580 abolished the enhancement of MMP-13, as well as collagenase-1 (MMP-1) and 92-kDa gelatinase (MMP-9) expression by TNF-(alpha) and TGF-(beta). Blocking the ERK1, 2 pathway by PD 98059 had no effect on the induction of MMP-13 expression by TNF-(alpha) or TGF-(beta), but potently suppressed MMP-1 and MMP-9 production. Inhibition of p38 activity by SB 203580 also suppressed collagenolytic activity produced by both cell lines and inhibited invasion of TNF-(alpha) or TGF-(beta) stimulated A-5 cells through type I collagen and reconstituted basement membrane (Matrigel). These results show that activation of p38 MAPK pathway plays a crucial role in the invasive phenotype of transformed squamous epithelial cells, suggesting p38 MAPK as a target to specifically inhibit their invasion.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Constitutive expression of G-CSF and GM-CSF in human skin carcinoma cells with functional consequence for tumor progression.

Tumor progression is characterized by an increasing escape of tumor cells from the growth control of their microenvironment, often caused by aberrant expression of growth factors. In the human skin carcinoma model system, based on the HaCaT keratinocyte line, tumor progression to high-grade malignant cells is associated with constitutive expression and secretion of the hematopoietic growth factors G-CSF and GM-CSF in vitro and in vivo. All HaCaT keratinocyte variants express the G-CSF and the GM-CSF receptors at levels comparable to normal keratinocytes. Consequently, they exhibit a stimulation of cell proliferation and migration in culture when treated with these factors. Moreover, both proliferation and migration of the high-grade malignant cells were strongly inhibited by neutralizing antibodies to G-CSF and GM-CSF, respectively. This demonstrates the functional role of these factors in high-grade malignant HaCaT cells through an autocrine mechanism in vitro and implies their significance in tumor progression in vivo. In light of the increasing use of G-CSF and GM-CSF in adjuvant tumor therapy, our data, as well as those discussed for head-and-neck tumors and gliomas, warrant a careful re-evaluation of the clinical application of both factors.

Animals↗

PubMed1999

Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature.

UV-radiation is a major risk factor for non-melanoma skin cancer causing specific mutations in the p53 tumor suppressor gene and other genetic aberrations. We here propose that elevated temperature, as found in sunburn areas, may contribute to skin carcinogenesis as well. Continuous exposure of immortal human HaCaT skin keratinocytes (possessing UV-type p53 mutations) to 40 degrees C reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors. Growth at 40 degrees C was correlated with the appearance of PARP, an enzyme activated by DNA strand breaks and the level corresponded to that seen after 5 Gy gamma-radiation. Concomitantly, comparative genomic hybridization (CGH) analyis demonstrated that chromosomal gains and losses were present in cells maintained at 40 degrees C while largely absent at 37 degrees C. Besides individual chromosomal aberrations, all tumor-derived cells showed gain of chromosomal material on 11q with the smallest common region being 11q13.2 to q14.1. Cyclin D1, a candidate gene of that region was overexpressed in all tumor-derived cells but cyclinD1/cdk4/cdk6 kinase activity was not increased. Thus, these data demonstrate that long-term thermal stress is a potential carcinogenic factor in this relevant skin cancer model, mediating its effect through induction of genetic instability which results in selection of tumorigenic cells characterized by gain of 11q.

Aneuploidy↗

PubMed1999

Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization.

The development of skin carcinomas presently is believed to be correlated with mutations in the p53 tumor suppressor and ras gene as well as with the loss of chromosome 9. We now demonstrate that, in addition, loss of chromosome 15 may be a relevant genetic defect. Reintroduction of an extra copy of chromosome 15, but not chromosome 4, into the human skin carcinoma SCL-I cells, lacking one copy of each chromosome, resulted in tumor suppression after s.c. injection in mice. Transfection with thrombospondin-1 (TSP-1), mapped to 15q15, induced the same tumor suppression without affecting cell proliferation in vitro or in vivo. Halted tumors remained as small cysts encapsulated by surrounding stroma and blood vessels. These cysts were characterized by increased TSP-1 matrix deposition at the tumor/stroma border and a complete lack of tumor vascularization. Coinjection of TSP-1 antisense oligonucleotides drastically reduced TSP-1 expression and almost completely abolished matrix deposition at the tumor/stroma border. As a consequence, the tumor phenotype reverted to a well vascularized, progressively expanding, solid carcinoma indistinguishable from that induced by the untransfected SCL-I cells. Thus, these data strongly suggest TSP-1 as a potential tumor suppressor on chromosome 15. The data further propose an unexpected mechanism of TSP-1-mediated tumor suppression. Instead of interfering with angiogenesis in general, in this system TSP-1 acts as a matrix barrier at the tumor/stroma border, which, by halting tumor vascularization, prevents tumor cell invasion and, thus, tumor expansion.

Animals↗

PubMed1999

12-O-tetradecanoylphorbol-13-acetate induces clonal expansion of potentially malignant keratinocytes in a tissue model of early neoplastic progression.

Tumor promoters stimulate the selective expansion of initiated mouse keratinocytes in the two-stage model of skin carcinogenesis. However, it is not clear whether these promoters directly modulate the growth of initiated cells or rather permit clonal expansion of initiated cells by modifying the environment of adjacent normal cells. The goal of this study was to further understand the mechanism of action of tumor promotion during early neoplastic progression of human stratified epithelium. To accomplish this, we have established an organotypic culture model that mimics a preneoplastic tissue and contains mixtures of genetically marked (beta-galactosidase), low-grade malignant keratinocytes (HaCaT-ras II-4) and normal human keratinocytes (NHKs) to monitor the fate and phenotype of these cells after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In submerged culture, concentrations of 0.001-1 microg/ml TPA were shown to limit the growth of NHKs yet had no effect on growth of II-4 cells. TPA (0.001 microg/ml) was then added to organotypic cultures containing mixtures of NHK:II-4 cells at varying ratios to determine whether this agent could selectively stimulate clonal expansion of II-4 cells in a normal epidermal background. Immunofluorescence for beta-galactosidase demonstrated that TPA caused a significant increase in the percentage of beta-galactosidase-positive areas in 12:1 and 4:1 mixtures. This TPA-induced expansion of II-4 cells was associated with a marked decrease in proliferation of NHKs, suggesting that II-4 could selectively expand because of its growth advantage relative to NHKs. Clonal expansion of tumor cells was temporally linked to the decreased expression of filaggrin and keratin 1 expression in adjacent NHKs. These findings indicate that TPA may enable expansion of potentially malignant cells through the epigenetic modification of proliferation in NHKs and differentiation of NHK and II-4 cells.

Animals↗

PubMed1999

Autocrine growth regulation by granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor in human gliomas with tumor progression.

Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) and/or their receptors are increasingly detected in solid human tumors, although little is known about their function in tumor growth and invasion. We analyzed RNA and protein expression of both factors and their receptors in 22 human gliomas (WHO grade II, III, and IV) and derived cell cultures. G-CSF, GM-CSF, and/or their receptors were expressed in all tumors and derived cell cultures, but coexpression of both factors and receptors was almost exclusively found in grade IV glioblastomas and thus correlated with advanced tumor stage. The functional significance of G-CSF and GM-CSF as regulators for glioma cells was demonstrated by 1) stimulation of proliferation and migration in tumor cells expressing one or both receptors by the corresponding factor; 2) inhibition of growth and migration of glioma cells expressing G-CSF, GM-CSF, and their receptors by neutralizing antibodies to both factors. These results indicate a significant role for both factors in the autocrine regulation of growth and migration in late-stage malignant gliomas and suggest a shift from paracrine to autocrine regulation with tumor progression. The implication of G-CSF and GM-CSF in glioblastoma growth regulation could make these factors further prognostic indicators and raises questions concerning their use in cancer therapy.

Autocrine Communication↗