Basal cell carcinomas without histological confirmation and their treatment: an audit in four European regions.

BACKGROUND: Limited data are available on how often basal cell carcinomas (BCCs) are clinically diagnosed without histological confirmation and how they are treated. OBJECTIVES: Within the framework of the EPIDERM project, an audit was conducted in four European countries to study the occurrence of clinically diagnosed BCCs without histological confirmation and to investigate how these are treated. METHODS: In the Netherlands, Scotland, Finland and Malta studies were performed within different timeframes. Patients with one or more BCC(s) were selected and the number of clinically diagnosed BCCs without histological confirmation and their treatment was investigated by (manually) reviewing the (electronic) patient records and checking the (hospital) pathology databases to find evidence of histological confirmation. RESULTS: In the Netherlands, 1089 patients with a first histologically confirmed BCC developed 1974 BCCs of which 1833 (92·9%) were histologically confirmed and 141 (7·1%) were not. A 4-month retrospective study conducted in Scotland selected 294 patients with 344 BCCs; 306 (89·0%) were histologically confirmed and 38 (11·0%) were not. A 3-month prospective study performed at the same centre in Scotland identified 44 patients who developed 58 BCCs; 44 (75·9%) of these were histologically confirmed and 14 (24·1%) were not. In Finland, there were 701 patients who developed 977 BCCs, of which 807 (82·6%) were histologically and 170 (17·4%) nonhistologically confirmed. In Malta, there were 420 patients with 477 BCCs. Only three (0·7%) of them were clinically diagnosed without histological confirmation. In the Netherlands and Finland, clinically diagnosed BCCs without histological confirmation were most often treated with cryotherapy, whereas in Scotland 5% imiquimod cream was the preferred treatment modality. CONCLUSIONS: Although the frequency of clinically diagnosed BCCs without histological confirmation differed between the four European regions (range 0·7-24·1%), this confirms that the burden of BCC in Europe is underestimated when based on data from pathology and/or cancer registries.

Assessing physicians' preferences on skin cancer treatment in Europe.

BACKGROUND: A wide variety of both surgical and nonsurgical therapies is currently available for patients with skin cancer. OBJECTIVES: This part of the EPIDERM (European Prevention Initiative for Dermatological Malignancies) project is aimed at the evaluation of the treatment preferences for skin cancer in eight countries of the European Union. METHODS: A multicentre hospital-based case-control study was carried out at dermatology departments in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain. Patients with skin cancer (basal cell carcinoma, actinic keratosis, squamous cell carcinoma, cutaneous malignant melanoma and Bowen disease) were consecutively enrolled between July 2008 and July 2010. Information on the study variables (sex, age, country, tumour type, anatomical location and treatment) was obtained from questionnaires designed by the EPIDERM project. RESULTS: In total, 1708 patients with skin cancer were included. Surgery was the first treatment option in 76·5% of the patients (P = 0·001). Actinic keratosis was the only tumour type in which nonsurgical treatment was more frequent than surgery (91·4%). Tumours on the head were less likely to be surgically excised than those at other locations (odds ratio 0·25, P = 0·001). Simple excision or curettage was the most common surgical procedure (65·4%), followed by graft and flaps (22·4%). Cryotherapy was the most common nonsurgical option (52·4%), followed by imiquimod (18·0%), photodynamic therapy (PDT; 12·0%), 5-fluorouracil (5-FU; 5·7%), and diclofenac with hyaluronic acid (4·0%). CONCLUSIONS: Surgery remains the first-choice treatment of skin cancer. Regarding nonsurgical treatments, the conservative treatments available (imiquimod, 5-FU, PDT and diclofenac gel) have not yet exceeded the use of ablative options such as cryotherapy despite their accepted benefit of treating field cancerization.

Known and potential new risk factors for skin cancer in European populations: a multicentre case-control study.

BACKGROUND: During recent years numerous studies have suggested that personal and environmental factors might influence cancer development. OBJECTIVES: To investigate environmental and personal characteristics associated with skin cancer risk. METHODS: A multicentre hospital-based case-control study was performed in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain, including 409 patients with squamous cell carcinoma (SCC), 602 with basal cell carcinoma (BCC) and 360 with cutaneous malignant melanoma (CMM) and 1550 control persons. Exposures were assessed by questionnaires that were partly self-administered, partly completed by dermatologists. Unconditional logistic regression modelling was used to assess associations including the influence of certain drugs and food items on skin cancer risk. RESULTS: The usual associations were observed for sun exposure and pigmentation characteristics, with chronic sun exposure being most strongly associated with SCC risk, and naevi and atypical naevi with CMM risk. Use of ciprofloxacin was associated with a decreased risk of BCC [odds ratio (OR) 0·33] and use of thiazide diuretics was associated with an increased risk of SCC (OR 1·66). Ciprofloxacin was also associated with SCC (OR 0·34) and thiazines with BCC (OR 2·04), but these associations lost significance after correction for multiple testing. Consumption of pomegranate, rich in antioxidants, was associated with decreased BCC and SCC risk, also after correcting for multiple testing. Recent experience of stressful events was associated with increased risk, particularly of CMM. CONCLUSIONS: In this large case-control study from across Europe the expected associations were observed for known risk factors. Some new potential protective factors and potential risk factors were identified for consumption of certain food items, medication use and stress, which deserve further investigation in future studies.

Risk factors for actinic keratosis in eight European centres: a case-control study.

BACKGROUND: There are limited data regarding the association of actinic keratosis (AK) and other types of nonmelanoma skin cancer (NMSC); studies investigating possible correlation of AK with melanocytic naevi are even scarcer. To our knowledge, there are no data examining the risk of AK in people using specific medications. OBJECTIVE: To investigate constitutional and exposure risk factors leading to AK and the coexistence of AK with NMSC and melanoma. METHODS: A multicentre hospital-based case-control study was performed in Finland, Germany, Greece, Italy, Malta, Poland, Scotland and Spain, including 343 patients with actinic keratosis (AK), 409 with squamous cell carcinoma (SCC), 602 with basal cell carcinoma (BCC), 360 with invasive melanoma and 119 with in situ melanoma, and 686 control subjects. Exposures were assessed by questionnaires that were partly self-administered and partly filled out by dermatologists. Unconditional logistic regression modelling was used to assess associations including the influence of phenotypic characteristics, presence of naevi, sun-exposure habits and certain drugs on AK risk. RESULTS: Differences in hair and eye coloration variably influenced the risk for AK, with red hair signifying a seven times higher risk [odds ratio (OR) 6·9, 95% confidence interval (CI) 4·34-11·00), and brown - compared with blue - eyes, about a 40% reduced risk (OR 0·61, 95% CI 0·13-0·92). The darker the skin phototype, the lower the risk for AK, with phototype IV exhibiting nine times less risk of developing AK. Some and many freckles on the arms were associated with an OR of 1·8 (95% CI 1·08-2·81) and 3·0 (95% CI 1·10-3·54), respectively, while overall number of naevi and high educational level were inversely associated with AK. Sun exposure, thiazide diuretics and cardiac drugs had a higher risk for AK. SCC was the most frequent (58%) skin neoplasm coexisting with AKs, followed by BCC (30%), melanoma in situ (12%) and invasive melanoma (6%). CONCLUSION: In this large case-control study from across Europe the expected associations were confirmed for known risk factors. Some possible new risk factors, including cardiac and diuretic drugs, were identified, creating a new field for further investigation in future studies.

The patient journey: a report of skin cancer care across Europe.

BACKGROUND: There are poorly documented variations in the journey a skin cancer patient will follow from diagnosis to treatment in the European Union. OBJECTIVES: To investigate the possible difficulties or obstacles that a person with a skin malignancy in the European Union may have to overcome in order to receive adequate medical screening and care for his/her condition. In addition, we wished to explore differences in European health systems, which may lead to health inequalities and health inequities within Europe. METHODS: Ten European countries took part in this investigation (in alphabetical order): Finland, Germany, Greece, Italy, Malta, Poland, Romania, Spain, the Netherlands and the U.K. The individual participants undertook local and national enquiries within their own country and completed a questionnaire. RESULTS: This exercise has identified important differences in the management of a skin cancer patient, reflecting major disparities in health care between European countries. CONCLUSIONS: Further investigation of health disparities and efforts to address health inequalities should lead to improvements in European health care quality and reduction in morbidity from skin cancer.

Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity.

Cultured bovine capillary endothelial (BCE) cells synthesize heparan sulfate proteoglycans (HSPG), which are both secreted into the culture medium and deposited in the cell layer. The nonsoluble HSPGs can be isolated as two predominant species: a larger 800-kD HSPG, which is recovered from preparations of extracellular matrix, and a 250-kD HSPG, which is solubilized by nonionic detergent extraction of the cells. Both HSPG species bind bFGF. 125I-bFGF bound to BCE cell cultures is readily released by either heparinase or plasmin. When released by plasmin, the growth factor is recovered from the incubation medium as a complex with the partly degraded high molecular mass HSPG. Endogenous bFGF activity is released by a proteolytic treatment of cultured BCE cells. The bFGF-binding HSPGs are also released when cultures are incubated with the inactive proenzyme plasminogen. Under such experimental conditions, the release of the extracellular proteoglycans can be enhanced by treating the cells either with bFGF, which increases the plasminogen activating activity expressed by the cells, or decreased by treating the cells with transforming growth factor beta, which decreases the plasminogen activating activity of the cells. Specific immune antibodies raised against bovine urokinase also block the release of HSPG from BCE cell cultures. We propose that this plasminogen activator-mediated proteolysis provides a mechanism for the release of biologically active bFGF-HSPG complexes from the extracellular matrix and that bFGF release can be regulated by the balance between factors affecting the pericellular proteolytic activity.

Keratinocyte growth factor is a bifunctional regulator of HPV16 DNA-immortalized cervical epithelial cells.

Various factors are known to regulate cell growth and differentiation in epithelial-mesenchymal interactions. Keratinocyte growth factor (KGF), an epithelial-specific cytokine produced by dermal fibroblasts and other mesenchymal cells, appears to affect growth, migration, and differentiation in epithelial-mesenchymal interactions. We have previously shown that human embryonic skin fibroblasts induce anchorage-independent growth of HPV16 DNA-immortalized human uterine exocervical epithelial cells (HCE16/3 cell line) in cocultures of HCE16/3 cells and fibroblasts. Here we report that KGF may be a major factor influencing growth and behavior of HCE16/3 cells in the coculture system. KGF stimulated both DNA synthesis and proliferation of normal human cervical epithelial (HCE) cells and HCE16/3 cells and the increase was stronger in HCE16/3 cells than in HCE cells. SiHa cells, a cervical carcinoma cell line with integrated HPV16 DNA, did not respond to the KGF mitogen signal. KGF receptor (KGFR) studies suggested that the different responses to the KGF mitogen signal may be correlated with KGFR. In addition, KGF alone was able to induce anchorage-independent growth of HCE16/3 cells, suggesting a potential role for KGF in the transformation process of epithelial cells. However, the transcription of HPV16 early genes was suppressed by KGF in the immortalized HCE16/3 cells, and this appeared to be due to transcriptional repression rather than a posttranscriptional process according to nuclear run-on analysis. In contrast, viral gene expression was not affected by KGF in SiHa cells. Our results suggest that KGF is a bifunctional growth factor in the HPV-immortalized cells, a positive regulator of cell growth and negative regulator of HPV16 early gene expression.

The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells.

Basic fibroblast growth factor (bFGF), a potent inducer of angiogenesis in vivo, stimulates the production of both urokinase- and tissue-type plasminogen activators (PAs) in cultured bovine capillary endothelial cells. The observed increase in proteolytic activity induced by bFGF was effectively diminished by picogram amounts of transforming growth factor beta (TGF beta), but could not be abolished by increasing the amount of TGF beta. However, the inhibition by TGF beta was greatly enhanced if the cells were pretreated with TGF beta before addition of bFGF. After prolonged incubation of cultures treated simultaneously with bFGF and TGF beta, the inhibitory effect of TGF beta diminished and the stimulatory effect of the added bFGF dominated as assayed by PA levels. TGF beta did not alter the receptor binding of labeled bFGF, nor did a 6-h pretreatment with TGF beta reduce the amount of bFGF bound. The major difference between the effects of bFGF and TGF beta was that while bFGF effectively enhanced PA activity expressed by the cells, TGF beta decreased the amounts of both cell-associated and secreted PA activity by decreasing enzyme production. Both bFGF and TGF beta increased the secretion of the endothelial-type plasminogen activator inhibitor.

Enhanced production and extracellular deposition of the endothelial-type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta.

Cultured human embryonic lung fibroblasts were used as a model to study the effects of transforming growth factor-beta (TGF beta) on the plasminogen activator (PA) activity released by nontumorigenic cells into the culture medium. The cells were exposed to TGF beta under serum-free conditions, and the changes in PA activity and protein metabolism were analyzed by caseinolysis-in-agar assays, zymography, and polypeptide analysis. Treatment of the cells with TGF beta caused a significant decrease in the PA activity of the culture medium as analyzed by the caseinolysis-in-agar assays. The quantitatively most prominent effect of TGF beta on confluent cultures of cells was the induction of an Mr 47,000 protein, as detected by metabolic labeling. The Mr 47,000 protein was a PA inhibitor as judged by reverse zymography. It was antigenically related to a PA inhibitor secreted by HT-1080 tumor cells as demonstrated with monoclonal antibodies. The induced Mr 47,000 inhibitor was deposited into the growth substratum of the cells, as detected by metabolic labeling, immunoblotting analysis, and reverse zymography assays of extracellular matrix preparations. TGF beta also decreased the amounts of urokinase-type and tissue-type PAs accumulated in the conditioned medium, as detected by zymography. Epidermal growth factor antagonized the inhibitory effects of TGF beta by enhancing the amounts of the PAs. These results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.

Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation.

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells.

We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogeneous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAI-1, we stained the cells live at 0 degree C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10(-6) M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PA-mediated focal proteolysis.

Short incubation with methyl aminolevulinate for photodynamic therapy of actinic keratoses.

BACKGROUND: Photodynamic therapy (PDT) using methyl aminolevulinate (MAL) is an effective first-line treatment for actinic keratoses. A reduced incubation period may have practical advantages. OBJECTIVE: This study aims to evaluate the effect of incubation time (1 vs. 3 h), MAL concentration (160 mg/g vs. 80 mg/g) and lesion preparation in the setting of MAL-PDT for treatment of actinic keratosis (AK). DESIGN: Open, randomized, parallel-group multicentre study. SETTING: Outpatient dermatology clinics. SUBJECTS: One hundred and twelve patients with 384 previously untreated AK. Most lesions (87%) were located on the face and scalp and were thin (55%) or moderately thick (34%). METHODS: Lesions were debrided, and MAL cream (160 mg/g or 80 mg/g) was applied before illumination with red light (570-670 nm; light dose, 75 J/cm2). Patients were followed up at 2 and 3 months. Sixty patients (54%) were re-treated and assessed at 6 months. MAIN OUTCOME: Complete lesion response rates 3 and 12 months after last treatment. RESULTS: For lesions on the face/scalp, lesion complete response rates were 78% for thin AK and 74% for moderately thick AK lesions after 1 h vs. 96% and 87% after 3 h incubation with MAL 160 mg/g. Lesion recurrence rates at 12 months after two treatments were similar [19% (3 of 16) with 1 h vs. 17% (3 of 18) with 3 h 160 mg/kg MAL-PDT] and lower than for 80 mg/g MAL-PDT (44-45%). CONCLUSION: MAL-PDT using a 1-h incubation may be sufficient for successful treatment of selected AK lesions.

Blood-derived gene-expression profiling in unravelling susceptibility to recessive disease.

Identification of new disease predisposition genes with chip-based technologies typically requires extensive financial and sample resources. We have recently shown that combining peripheral blood genome and transcriptome (BGT) information in highly selected materials can be a successful low-cost approach to unravelling dominant tumour susceptibility. In this study, we extended our investigations to recessively inherited tumour predisposition, and identified a homozygous germline mutation in the damage-specific DNA binding protein 2 (DDB2) gene in a patient with several facial tumours, for which doctors had been unable to provide a diagnosis. Our results provide proof of principle that BGT is a powerful approach for both dominant and recessive genes. In addition to tumour susceptibility, the method may be useful in characterising genetic defects underlying other disease phenotypes.

Synthesis and secretion of alpha-2-macroglobulin by cultured adherent lung cells. Comparison with cell strains derived from other tissues.

We studied the synthesis and secretion of alpha-2-macroglobulin by cultures of human adherent cells. Much more alpha-2-macroglobulin (measured by radioimmunoassay) accumulated in media of established strains of adherent cells derived from embryonic lung than in media of established strains derived from adult skin or rheumatoid synovium. Alpha-2-macroglobulin accumulated in media of primary cultures of adherent cells from a variety of embryonic tissues. However, the amount of alpha-2-macroglobulin accumulating in media of subsequent passage of these cells declined for all strains except those derived from lung. Immunodiffusion and double-antibody immunoprecipitation studies of cell extracts and media after incubation of cells with l-[(35)S]methionine supported the radioimmunoassay finding that adherent cells from lung synthesized and secreted more alpha-2-macroglobulin than adherent cells from skin. Intracellular alpha-2-macroglobulin could not be detected by radio-immunoassay or visualized by immunofluorescent microscopy, suggesting that synthesized alpha-2-macroglobulin is rapidly secreted. Plasminogen-rich fibrin clots were lysed in culture media of adherent cells from embryonic lung and, to a lesser extent, heart. Adherent cells from other tissues, which produced less alpha-2-macroglobulin, did not lyse fibrin clots. However, all cultures of adherent cells contained pericellular fibronectin, a large, external, transformation-sensitive glycoprotein known to be cleaved by plasmin. We speculate that production of alpha-2-macroglobulin may be a means for protease-secreting normal cells to preserve cell surface integrity and that alpha-2-macroglobulin synthesized locally in lung may protect lung tissues from a variety of proteases.

Presence of alpha-2-macroglobulin in normal but not in malignant cervical epithelium.

alpha-2-Macroglobulin (alpha 2M) was demonstrated in normal and mildly dysplastic cervical epithelium using immunohistochemical methods. In contrast, staining for alpha 2M diminished gradually with advancing epithelial dysplasia and was totally negative in truly neoplastic lesions. No in vitro synthesis could be detected in cultured ectocervical cells or in a corresponding malignant cell line (HeLa). However, alpha 2M was taken up from added human serum by cultured normal ectocervical cells, whereas HeLa cells remained negative also under these conditions. These and previous results suggest that normal cells may have the capacity to synthesize or endocytose alpha 2M, thus possibly regulating their pericellular proteolysis, while these functions may be defective in malignantly transformed cells.

Regulation of plasminogen activator activity in human fibroblastic cells by fibrosarcoma cell-derived factors.

Low-molecular-weight protein factors (Mr 8,000 to 18,000) from serum-free conditioned medium of human fibrosarcoma (8387) cells reversibly enhanced the secretion of proteinase-inhibitory activity by cultured normal human skin fibroblasts. This inhibitory activity could be absorbed by immobilized plasminogen activator (PA) of urokinase type but not by heparin, and it was sensitive to treatment with sodium dodecyl sulfate. The secretion of a heparin-binding Mr 60,000 proteinase inhibitor, resembling protease nexin, was also detected. Early passages of adult skin fibroblasts do not contain or secrete PA. When cell types secreting this enzyme were tested, the fibrosarcoma-derived factors decreased the PA secretion detectable after sodium dodecyl sulfate treatment in all conditioned media of normal and malignant fibroblastic cells examined, including the 8387 cell line itself. However, no effects on the secretion of PA by normal or malignant cells of epithelioid origin or by melanoma cells were seen. A similar preparation from human epidermoid carcinoma (A431)-conditioned medium did not affect the PA activity or secretion of proteinase inhibitors from fibroblastic cells. The ability of sarcoma cells to modulate the production of PA inhibitors is a novel characteristic in the regulation of cellular proteolysis.

Presence of alpha 2-macroglobulin in normal but not in malignant human syncytiotrophoblasts.

alpha 2-Macroglobulin (alpha 2M) was demonstrated in normal syncytiotrophoblasts of both early and full-term human placentas using immunocytological staining. alpha 2M was also detected in hydatidiform moles, the benign tumors of proliferating syncytiotrophoblasts. In contrast, no alpha 2M was detected in invasive moles or choriocarcinomas. In culture conditions, both normal syncytiotrophoblasts and choriocarcinoma cells, identified by production of human chorionic gonadotropin, were negative when stained for alpha 2M or when studied using metabolic labeling and immunoprecipitation or radioimmunoassay. However, alpha 2M was taken up from added human serum by the cultured syncytiotrophoblasts, whereas choriocarcinoma cells remained negative also under these conditions. The possible role of alpha 2M in the regulation of proteolysis in cell invasion is considered.

Human ornithine decarboxylase-overproducing NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice.

Overexpression of human ornithine decarboxylase (ODC) under the control of strong promoters induces morphological transformation of immortalized NIH3T3 and Rat-1 fibroblasts [M. Auvinen et al., Nature (Lond.), 360: 355-358, 1992]. We demonstrate here that ODC-overproducing NIH3T3 cells are tumorigenic in nude mice, giving rise to rapidly growing, large fibrosarcomas at the site of inoculation. The tumors are capable of invading host fat and muscle tissues and are vascularized abundantly. To disclose the molecular mechanism(s) driving the tumorigenic, invasive, and angiogenic phenotype of the tumors, the ODC-overproducing cell lines and tumor tissues were analyzed for the expression of various potential regulators and mediators of cell proliferation, matrix degradation, and angiogenesis. The tumorigenicity of ODC transformants was associated with elevated polyamine levels and down-regulated growth factor receptors. The invasiveness of the ODC-induced tumors could not be attributed to overexpression of various known extracellular matrix-degrading proteases or matrix metalloproteinases. The induction of the tumor neovascularization proved not to be elicited by vascular endothelial growth factor or basic fibroblast growth factor. Instead, the ODC-overexpressing cells appeared to secrete a novel angiogenic factor(s) that was able to promote migration of bovine capillary endothelial cells in collagen gels and increase the proliferation of human endothelial cells in vitro. In parallel, ODC-transformed cells displayed down-regulation of thrombospondin-1 and -2, the negative regulators of angiogenesis. Thus, the induction of the angiogenic phenotype of the ODC transformants is likely due both to increased expression and secretion of the new angiogenesis-stimulating factor(s) and decreased production and release of the antiangiogenic thrombospondins.

Transforming growth factor beta1 induces apoptosis in normal melanocytes but not in nevus cells grown in type I collagen gel.

We used type I collagen gel cultures to compare the growth requirements of melanocytes and dermal nevus cells. Melanocytes but not nevus cells undergo apoptosis in collagen unless supplied with growth stimulators such as fibroblast growth factor 2. To characterize the mechanism of melanocyte apoptosis in collagen, we tested the effects of transforming growth factor beta1, known to be functionally active in the skin. When picomolar amounts of transforming growth factor beta1 were added to normal melanocytes grown in type I collagen gel, their apoptosis was dramatically accelerated. In contrast, the apoptotic rate of nevus cells and melanoma cells grown under similar conditions was not affected by transforming growth factor beta1. The increased apoptosis of normal melanocytes was effectively counteracted by addition of either neutralizing transforming growth factor beta1 antibodies or fibroblast growth factor 2 to the collagen gel. Interestingly, the background apoptosis of normal melanocytes was also inhibited by transforming growth factor beta1 antibodies. By Western blotting we detected transforming growth factor beta-like immunoreactivity in melanocyte, nevus cell, and melanoma cell lysates. A sensitive bioassay confirmed that their medium contained considerable amounts of heat-activatable growth inhibitory activity that could partly be neutralized by transforming growth factor beta1 antibodies. It is evident that apoptosis of melanocytes grown in type I collagen gel can be mediated by both endogenous and exogenous transforming growth factor beta. We suggest that the balance between inhibitory growth factors such as transforming growth factor beta and stimulatory growth factors like fibroblast growth factor 2 has the potential to regulate the growth, localization, and survival of normal melanocytes also in vivo. The resistance of nevus cells to transforming-growth-factor-beta-mediated apoptosis may facilitate their ability to grow in the dermal compartment of the skin.