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Ras-related C3 toxin substrate 1 (Rac1) is a small Rho-GTPase with important functions in fundamental cellular processes such as cytoskeleton rearrangements, signal transduction, cell cycle progression and malignant transformation. Using Rac1 primer, we identified a 5.5-kb DNA sequence on chromosome 4 (Chr. 4) in the human genome, containing the intronless protein coding sequence of Rac1. Sequence analysis revealed features of a processed pseudogene, which we named psi1Rac1, that could be detected by Southern blot and polymerase chain reaction (PCR) on genomic DNA. A psi1Rac1 pseudogene transcript was not detected by reverse transcription-polymerase chain reaction (RT-PCR), nor had the psi1Rac1 promoter any transcriptional activity. In addition, three other intronless pseudogenes of Rac1 on chromosomes 4, 13 and X were identified (psi1Rac1-psi4Rac1) sharing an 86-96% sequence similarity with Rac1. Neither RT-PCR with pseudogene specific restriction enzymes, nor the sequencing of 130 cDNA clones from benign and malignant breast tissue and cell lines, detected the transcription of any of the Rac1 pseudogenes (psi2Rac1-psi4Rac1). Existence of Rac1 pseudogenes should be taken into consideration when analyzing genomic alterations of the human Rac1 gene.

Positron emission tomographic (PET) investigations were performed to obtain in vivo information on symptomless radiation-induced pathological changes in the human spinal cord. PET investigations were carried out prior to radiotherapy and during the regular follow-up in an early hypopharyngeal cancer patient (the spinal cord was irradiated with a biologically effective dose of 80 Gy2), with [18F]fluorodeoxyglucose (FDG), [11C]methionine and [15O]butanol as tracers; radiosensitivity and electroneuronographic (ENG) studies were also performed. A very low background FDG accumulation (mean standardized uptake values, i.e. SUV: 0.84) was observed in the spinal cord before the initiation of radiotherapy. An increased FDG uptake was measured 2 months after the completion of radiotherapy (mean SUV: 1.69), followed by a fall-off, as measured 7 months later (mean SUV: 1.21). By 44 months after completion of irradiation, the FDG accumulation in the irradiated segments of the spinal cord had decreased to a level very close to the initial value (mean SUV: 1.11). The simultaneous [15O]butanol uptake results demonstrated a set of perfusion changes similar to those observed in connection with the FDG accumulation. The patient exhibited an extremely low [11C]methionine uptake within the irradiated and the nonirradiated spinal cord during the clinical course. She has not had any neurological symptoms, and the results of central ENG measurements before radiotherapy and 2 months following its completion proved normal. Radiobiological investigations did not reveal unequivocal signs of an increased radiosensitivity. A transitory increased spinal cord FDG uptake following radiotherapy may be related to the posttherapeutic mild inflammatory and regenerative processes. The normal [11C]methionine accumulation observed is strong evidence against intensive cell proliferation. The high degree of normalization of the temporarily increased FDG uptake of the irradiated spinal cord segments by 44 months is in good agreement with the results of monkey studies, which demonstrated a nearly complete recovery from radiation-induced spinal cord injury.

Purpose: To investigate the pathophysiology of the radiation-induced, chronic Lhermitte's sign (LS) on the basis of long-standing case histories with partial functional recovery., Patients and Methods: As radiotherapy in two nasopharyngeal cancer patients, a biologically effective dose (BED) of 103.8 Gy(2) (case 1) and 94.8 Gy(2) (case 2) was delivered to the cervical spinal cord. Neurologic signs relating to the irradiated spinal cord segments developed after 2 months (case 1) and 5 years (case 2), with radiation-induced damage equivalent to grade 3 (case 1) and grade 2 (case 2) toxicity (Common Toxicity Criteria, Version 2.0). The clinical status improved to grade 2 (case 1) and grade 1 (case 2). Positron emission tomography (PET) and fibroblast clonogen assay were applied 25 and 7 years postirradiation, respectively, to characterize this rare clinical picture., Results: PET demonstrated increased [(18)F]fluorodeoxyglucose (FDG) accumulation and [(15)O]butanol perfusion, but negligible [(11)C]methionine uptake in the irradiated spinal cord segments in both patients. In clonogenic assays, fibroblasts from case 1 displayed much higher radiation sensitivity than in healthy controls, while in case 2 the fibroblasts sensitivity was normal., Conclusions: These data suggests a close direct relationship between regional perfusion and metabolism of the spinal cord, similarly as in the brain. The postirradiation recovery may be related to energy-demanding conduction, explaining the increased metabolism and perfusion. The increased radiosensitivity and higher spinal cord BED may have contributed to the more severe sequelae in case 1.

Purpose: To study the efficacy of reirradiation as salvage treatment in patients with locally recurrent nasopharyngeal carcinoma., Patients and Methods: Between 1993 and 2000, 20 consecutive patients (twelve males and eight females) with nasopharyngeal cancer, previously irradiated in different Hungarian institutions, were reirradiated for biopsy-proven locally recurrent tumor. Histologically, 85% of the patients had WHO type III, 5% type II, and 10% type I disease. Stages I-IV (AJCC 1997 staging system) were assigned to five (25%), seven (35%), five (25%), and three (15%) patients, respectively; none of them had distant metastases, and only eight (40%) displayed regional dissemination. The median time period between termination of primary treatment and local recurrence was 30 (range, 10-204) months. Brachytherapy was the method most frequently used: in ten cases alone (especially for rT1 tumors), and in eight cases in combination with external beam therapy. Two patients with locally advanced disease underwent external beam therapy only. The median dose in the event of brachytherapy alone was 20 Gy (4 x 5 Gy or 5 x 4 Gy, range, 16-36 Gy), and the dose range for exclusive external irradiation was 30-40 Gy. In cases of combined irradiation, a median 20-Gy brachytherapy (range, 16-40 Gy) was associated with 30-40 Gy of external irradiation. Radiotherapy was supplemented by neck dissection (six patients), nasopharyngectomy (one patient), or chemotherapy (eleven patients)., Results: 16 patients were reirradiated once, three twice, and one patient three times, with a median equivalent dose for tumor effect of 36 Gy (mean, 44 Gy; range, 19-117 Gy; the estimated alpha/beta-ratio was 10 Gy). The median equivalent dose of reirradiation for late effect on normal tissue (with an estimated 70% delivery of the tumor dose) amounted to 30 Gy (mean, 37 Gy; range, 13-101 Gy, estimated alpha/beta-ratio 3 Gy). After a median follow-up of 37 (range, 12-72) months, the overall survival was 60% (12/20). Seven of the twelve surviving patients are currently tumor-free. After primary irradiation, xerostomy occurred in all patients as an unavoidable side effect of treatment. Following reirradiation, a severe (grade 3 or higher) late toxicity (CTC criteria, version 2) has been observed in two tumor-free patients (10%) so far (necrosis of soft palate and paresis of glossopharyngeal nerve)., Conclusion: Retreatment of nasopharyngeal carcinoma with radiotherapy (preferably a combined modality), can result in longterm local control and survival in a substantial proportion of patients, at the price of an acceptable morbidity.

Mucosal melanomas comprise about 1% of all malignant melanomas and exhibit far more aggressive behaviour than that of skin melanomas: they are more inclined to metastatize into regional and distant sites or recur locally, regionally or in distant locations, resulting in a high rate of cause-specific death. Mucosal melanomas in the head and neck region account for half of all mucosal melanomas, occurring mainly in the upper respiratory tract, oral cavity and pharynx. They appear with equal gender distribution and with a peak incidence in the age range 60-80 years. In consequence of their hidden location, they are usually diagnosed in a locoregionally advanced clinical stage, with a rate of 5-48% of regional and 4-14% of distant dissemination. The typical therapeutic approach is surgery, postoperative irradiation and systemic therapy. Local control with either surgery or radiotherapy is frequently (60- 70%) achieved, but the rates of local, regional and distant recurrences are high (50-90%, 20-60% and 30-70%, respectively). The reported 5-year actual survival rates are poor (17-48%), which is attributed mainly to a haematogenous dissemination. These characteristics demonstrate that identification of the precursor lesions and more effective local and systemic approaches are needed to improve the therapeutic results.

Radiation myelopathy is a rare, but extremely serious side-effect of radiotherapy. Recovery from radiation-induced motor sequelae is rare, whereas, the regeneration of sensory losses is relatively frequent. Among the sensory radiogenic injuries of the spinal cord, Lhermitte's sign (LS) is most frequent. This review describes the clinical picture and diagnostic imaging signs of radiogenic LS. There have been only a few studies on large patient groups with radiogenic LS, demonstrating a rate of occurrence of 3.6-13%, relating mainly to mantle irradiation or the radiotherapy of head and neck tumors. These cases typically manifest themselves 3 months following radiotherapy and gradually disappear within 6 months. Only 3 LS cases have been described in the English literature with extraordinarily severe symptoms lasting for more than 1 year. MRI, a sensitive tool in the detection of demyelination, failed to reveal any pathological sign accompanying radiogenic LS. However, positron emission tomography demonstrated increased [18F]fluorodeoxyglucose accumulation and [15O]butanol perfusion, but a negligible [11C]methionine uptake in the irradiated spinal cord segments in patients with long-standing LS. These imaging data are suggestive of a close direct relationship between the regional perfusion and metabolism of the spinal cord, very much like the situation in the brain. We postulate that an altered, energy-demanding conduction along the demyelinated axons of patients with chronic radiogenic LS may explain the increased metabolism and perfusion.

Objective: Authors analysed the prevalence of depressive symptomatology among the patients of a day time rehabilitation department for mobility disorders., Methods: Beck's Depression Inventory was used as a screening instrument. 57 subjects completed the inventory both at admission and discharge., Results: 33 subjects of 57 showed depressive symptoms at admission and 13 persons at discharge. The number of patients with mild depressive symptomatology decreased from 24 to 10, with medium level depression from 5 to 2 and with severe depression from 4 to 1. None of them have taken antidepressive drugs., Conclusions: These results confirm the high importance of the proper psychological conduction of patients with depressive symptoms.

Endothelial migration on extracellular matrix is regulated by integrins and proteolysis. Previous studies showed that beta(3)-integrins regulate expression of the urokinase-type plasminogen activator receptor (uPAR) through outside-in signalling involving the cytoplasmic domain. Here we show that overexpression of the integrin-binding protein beta(3)-endonexin decreased uPAR promoter (-398 base-pair fragment) activity that is constitutively active in endothelial cells. Mutation of the NF-kappaB promoter binding site (-45 bp) impaired the ability of beta(3)-endonexin to downregulate uPAR promoter activity. Immunoprecipitation studies showed that beta(3)-endonexin interacts directly with the p50/p65 transactivation complex and thereby inhibits binding of kappaB oligonucleotides to the p50/p65 complex. Moreover, binding of beta(3)-endonexin to p50 was inhibited in the presence of kappaB but not mutated kappaB oligonucleotides, suggesting a sterical competition between beta(3)-endonexin and kappaB DNA for the p50/p65 complex. We therefore propose that beta(3)-endonexin acts as regulator of uPAR expression in beta(3)-integrin-mediated endothelial cell migration through direct interaction with p50/p65. Since NF-kappaB regulates the expression of matrix degrading enzymes, the present results define a role of beta(3)-endonexin in regulating beta(3)-integrin-mediated adhesion and pericellular proteolysis.

Patients and Methods: In the period between 1993 and 2000, 20 patients with nasopharyngael cancer were re-irradiated for locally recurrent carcinomas. The median duration between primary treatment and recurrence was 30 months. Brachytherapy was the method most frequently used in 10 cases alone, and in 8 cases in combination with external beam therapy. 2 patients underwent only external beam therapy. The external irradiation was performed with CT/MRI-based treatment planning. Brachytherapy involved a high dose rate afterloading method. The cumulative dose of re-irradiation was 20-60 Gy., Results: After a median follow-up of 37 months the overall survival and the local control were 60% (12/20) and 58% (7/12), respectively. 7 of the 12 surviving patients are currently tumour-free. After primary irradiation xerostomy occurred in all patients as an unavoidable side-effect of the treatment. A severe (grade 3 or higher according to the Common Toxicity Criteria version 2.0) late-side effect has so far been observed in 2 cases (10%). Authors' results lie in the medial range of the data in the international literature, though the rate of radiogen side-effects in the patient group is low. Both results are assumed to be a consequence of the re-irradiation dose being lower than the value considered optimum in the recent literature., Conclusions: Re-irradiation of locally recurrent nasopharyngeal cancers is an efficient treatment modality, which should be used as a combination of external beam therapy and brachytherapy. The optimum cumulative dose is about 50-60 Gy. This dose results in a 5 years survival rate of about 40% with an acceptable (30%) risk. The results of re-irradiation may be improved, if PET is used to determine the extent of the surviving tumour tissue. This can help in the choice of the proper treatment modality.

Objective: To investigate the pathomechanism of the rare radiogenic lower motor neurone disease (LMND) on the basis of a case history involving a partial functional recovery., Patient: A 31-year-old seminoma patient received postoperative para-aortic and para-iliac telecobalt irradiation with a biologically effective dose of 88 Gy(2) (44 Gy in 2 Gy fractions/day, with an estimated alpha/beta of 2 Gy) delivered to the spinal cord following a single cycle of chemotherapy. LMND developed 4 months after the completion of radiotherapy. The patient exhibited flaccid paraparesis of the lower extremities (without sensory or vegetative signs), followed by a worsening after further chemotherapy, due to pulmonary metastatization. A gradual spontaneous functional improvement commenced and led several years later to a stabilized state involving moderately severe symptoms., Methods: In the 15th year of the clinical course, magnetic resonance imaging (MRI) and positron emission tomography (PET) with [(18)F]fluorodeoxyglucose (FDG) and [(11)C] methionine were conducted. Four lines of experiments (clonogenic assay using fibroblasts isolated from a skin biopsy sample of the patient, comet assay, micronucleus assay, and the testing of chromosome aberrations after in vitro irradiation of peripheral blood samples) were performed in a search for an increased individual radiosensitivity., Results: MRI investigations failed to reveal any pathological change. PET demonstrated an increased FDG accumulation, but a negligible [(11)C] methionine uptake in the irradiated spinal cord segments. The radiobiological investigations did not indicate any sign of an increased individual radiosensitivity., Conclusions: We suggest that the observed partial functional recovery and stabilization of the symptoms of radiogenic LMND may be explained by the higher than normal density of sodium channels expressed along the demyelinated axons of the restored conduction. The increased energy demands of this type of conduction are proved by a higher metabolic rate (increased FDG uptake) of the irradiated spinal cord segments without a substantial regenerative process (lack of detectable protein synthesis).

The authors have reviewed the financial considerations of oncological FDG PET examinations by the guidelines of the Health Care Financing Administration (USA). By critical assessment of large number of clinical investigations, the cost-effectiveness of FDG PET scans has been confirmed in the following cases: differential diagnosis of solitary pulmonary nodule, diagnosis, staging and restaging of non-small cell lung cancer, colorectal cancer, malignant lymphomas, melanoma malignum, esophageal neoplasms and cancers of the head and neck. The role of this method in breast cancer is currently under intensive investigation. Due to the correct staging, PET examinations in these indications enable the clinicians to choose the optimal treatment ensuring the maximum probability of recovery and being cost-effective as unnecessary medical interventions become avoidable.

Following the failure of conventional diagnostic procedures, whole-body FDG-PET investigations were carried out in 42 metastatic cancer patients to localize occult primary carcinomas. During the clinical follow-up, the presence of malignant tumor was ruled out in 3 cases, and 2 patients originally believed to have carcinoma were confirmed to be suffering from a malignant hematological disease. These false diagnoses were associated with the use of imaging methods only (2 cases) or cytology only (1 case), lack of double, independent pathological investigations (2 cases) or immunophenotyping (2 cases) and the occurrence of an unrecognized rare tumor in a hospital with a small patient turnover (1 case). The discovered 11 occult primaries (4 lung, 3 breast, 2 hypopharynx and 1 base of the tongue carcinomas and 1 non-Hodgkin lymphoma) led to a 28% diagnostic efficacy of PET (11/39 malignant pathological reports). This efficacy is doubled (60%) if PET reveals < or = 5 malignant lesions and the locations of the pathological foci are tumor-specific. We suggest PET investigations in the search for occult primaries following a controlled pathological diagnosis and the failure of conventional diagnostic procedures.

Whole-body FDG PET examinations in 10 cases of epipharyngeal tumour (8 males, 2 females, mean age: 48 years) have been performed since January 1999. The PET examinations were aimed at the accurate staging, follow-up of the patients after the treatment, identification of recurrencies and localization of the unknown primary tumor. Functional imaging resulted in "upstaging" in 3 patients as compared to staging by the conventional diagnostic tools. Four additional patients with hyperplastic epipharyngeal tissue were investigated for occult primary cancer after negative results of multiple excisions, resulting in one case of primary epipharyngeal cancer. Correct staging, early detection of recurrencies, localization of occult primary tumor and the better post-therapeutic assessment of epipharyngeal masses all facilitate a more reasonable therapeutic approach, which may improve survival results.

The authors have reviewed the financial considerations of oncological FDG PET examinations by the guidelines of the Health Care Financing Administration (USA). By critical assessment of large number of clinical investigations,the cost-effectiveness of FDG PET scans has been confirmed in the following cases: differential diagnosis of solitary pulmonary nodule, diagnosis,staging and restaging of non-small cell lung cancer, colorectal cancer, malignant lymphomas, melanoma malignum, esophageal neoplasms and cancers of the head and neck. The role of this method in breast cancer is currently under intensive investigation. Due to the correct staging, PET examinations in these indications enable the clinicians to choose the optimal treatment ensuring the maximum probability of recovery and being cost-effective as unnecessary medical interventions become avoidable.

Unlabelled: The relatively benign, but occasionally rapidly fatal clinical course of medullary thyroid cancer (MTC) has raised the need for individual survival probability estimation. A retrospective study on 91 MTC clinical case histories with a mean follow-up of 6 years indicated prevalences of local, regional and distant residual tumor on primary care completion of 23%, 54% and 54%, respectively. Local, regional and distant relapses during follow-up occurred in 8%, 23% and 26% of the patients, with a cause-specific death in 26% of the cases. Prognostic factors statistically significantly influencing the cause-specific survival were selected by uni- and multivariate analysis. A Markov method-based model was developed for the estimation of individual time-dependent local, regional and distant relapse-free and cause-specific survival probability functions, with parameters numerically determined via a maximum likelihood procedure. These parameters include relative risk factors related to prognosticators, a residual or recurrent local/regional/distant tumor, and combinations of these entities. In multivariate studies, the patient s age and gender, the genetic basis of the dis-ease, lymph node involvement, the existence of a general symptom (diarrhoea) at presentation, and the dosage of external irradiation proved to be prognosticators. The cause-specific survival function of the study population indicated mean 5, 10 and 15-year survival probabilities of 69%, 62% and 58%., Conclusion: Survival probabilities can be predicted for extrastudy cases provided that the same laws and principles govern the clinical course of these cases and those comprising the study. For individual survival probability estimation, a Pascal program (MEDUPRED) was written and is available on the home page of the National Institute of Oncology, Budapest (www.oncol.hu).

Gastric enterochromaffin-like cells produce histamine in response to the antral hormone gastrin and accumulate the biogenic amine in secretory organelles via vesicular monoamine transporter subtype 2. The putative effects of gastrin on vesicular monoamine transporter subtype 2 expression and promoter activity are poorly understood. In the present study we used highly enriched rat enterochromaffin-like cells (purity, >90%) and rat pheochromocytoma cells stably transfected with a gastrin/cholecystokinin B receptor to investigate the expression and transcriptional regulation of vesicular monoamine transporter subtype 2. Stimulation of vesicular monoamine transporter subtype 2 mRNA and protein expression was observed in isolated enterochromaffin-like cells after 3- to 7-h incubation with gastrin (10(-7) M), forskolin (10(-5) M), or ionomycin (10(-5) M). Deletion analysis of the rat vesicular monoamine transporter subtype 2 promoter defined the minimal promoter sequence necessary for full basal activity as a -121 bp segment upstream of exon 1 containing two Sp1 sites (-97 to -88 bp and -68 to -59 bp) and a cAMP-responsive element (-44 to -35 bp). Gastrin (10(-7) M) stimulated extracellular signal related kinase1/2 phosphorylation, activated Sp1 and cAMP-responsive element-binding protein, and further induced activity of the complete rat vesicular monoamine transporter subtype 2 promoter (-800 bp) in gastrin/cholecystokinin B receptor cells. The -121-bp fragment was able to confer full gastrin responsiveness, and site-directed mutagenesis of the Sp1 and cAMP-responsive element motifs demonstrated their crucial importance for basal and inducible activities. Comparison of promoter activity of histidine decarboxylase, chromogranin A, or vesicular monoamine transporter subtype 2 in transfected cell lines revealed significant differences in basal and gastrin-stimulated activities. Our current study provides the first evidence that gastrin directly stimulates the expression and promoter activity of vesicular monoamine transporter subtype 2. Sp1 and cAMP-responsive element-binding protein recognition motifs located within 121 bp upstream of exon 1 appear to be indispensable for full basal and inducible promoter activities. Diverging effects of gastrin on histidine decarboxylase, chromogranin A, and vesicular monoamine transporter subtype 2 promoter may account for the coordinated synthesis and storage of histamine in this neuroendocrine cell type.

Objective: Matrix metalloproteinases (MMPs) are frequently expressed in malignant tumors and play an important role in tumor invasion and metastasis. MMP-2 and MMP-9 expression has been correlated with poor survival in some tumors, but data for ovarian cancer are lacking, despite clinical trials with MMP inhibitors. The aim of this study was to assess activity of MMP-2 and MMP-9 and correlate it to prognosis in ovarian cancer., Methods: MMP-2 and MMP-9 gelatinolytic activity was analyzed in 84 patients with advanced ovarian cancer FIGO stage III and 19 benign ovarian tumors by gelatin zymography. MMP-9 immunoreactivity was detected by immunohistochemistry and gelatinolytic activity was localized in ovarian cancer tissue by in situ zymography., Results: were correlated with patient survival, with a median follow-up period of 55 months. Results. Median pro-MMP-9 activity was at 0.00 U/microg protein in benign ovarian tissues and 4.82 U/microg protein in ovarian cancer (P = 0.001); activated MMP-9 was not detected. Pro-MMP-2 expression in benign ovarian tissue did not differ from that of malignant ovarian tissue, whereas active MMP-2 was present in 52% of ovarian cancers, but absent in benign ovarian tissues. Analyzing all patients high pro-MMP-9 activity was associated with short overall survival (P = 0.019) while pro-MMP-2 and activated MMP-2 did not predict overall survival. When analyzing the subgroups of patients with and without residual tumor mass at the time of surgery, pro-MMP-9 was of prognostic value only in the subgroup of patients with no residual tumor mass. In univariate analysis pro-MMP-9 activity, residual tumor mass, age, ascites volume, and grading were of prognostic significance for overall survival. However, in multivariate analyses, including all biological and clinicopathologic variables, only pro-MMP-9 and residual disease remained statistically independent prognostic factors. In situ zymography localized gelatinolytic activity predominantly to the tumor cell nests displaying MMP-9 immunoreactivity., Conclusions: Pro-MMP-9 gelatinolytic activity, but not active MMP-2 or MMP-9, serves as a useful statistically independent prognostic factor in ovarian cancer FIGO stage III, thus helping to identify ovarian cancer patients with an aggressive form of the disease., (Copyright 2001 Academic Press.)

Proteases are linked to the malignant phenotype of different solid tumors. Therefore, the expression of the matrix metalloproteinase (MMP)-2 and MMP-9 and of the serine protease urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) in the progression of ovarian cancer was investigated. Gelatinolytic activity and protein expression of MMP-2 and MMP-9 were analyzed in tissue extracts of 19 cystadenomas and 18 low malignant potential (LMP) tumors, as well as 41 primary tumors of advanced ovarian cancer stage International Federation of Gynecology and Obstetrics IIIc/IV and their corresponding omentum metastases by quantitative gelatin zymography and Western blot. In the same tissue extracts, antigen levels of uPA and its inhibitor PAI-1 were determined by ELISA. Protein expression of pro-MMP-2 (72 kDa) and pro-MMP-9 (92 kDa as well as antigen levels of uPA and PAI-1 were low in benign ovarian tumors but increased significantly from LMP tumors to advanced ovarian cancers. The highest values of all of the proteolytic factors were detected in omentum metastases. Active MMP-2 enzyme (62 kDa) was detected only in ovarian cancer (66%) and corresponding metastases (93%) but never in benign or LMP tumors. The activation rate of MMP-2 to its active isoform was higher in the metastases. Comparing both proteolytic systems, higher PAI-1 concentrations were consistently found in cancers with high pro-MMP-9 expression. These data indicate that members of the plasminogen activator system, as well as the metalloproteinases MMP-2/9, increase with growing malignant potential of ovarian tumors. These findings are of particular relevance to the development of protease inhibitors as new therapeutic approaches in ovarian cancer.

The urokinase type plasminogen activator (uPA), together with its receptor uPAR and the plasminogen activator inhibitor type-1 (PAI-1) plays a pivotal role during tumor invasion and metastasis. Integrins, via interaction with the extracellular matrix (ECM), control cell adhesion and motility. The two systems are functionally linked because uPAR and PAI-1 bind to the ECM component vitronectin (VN). Because integrin signaling alters gene expression patterns, we investigated whether the expression levels of uPA, uPAR, and PAI-1 are affected by ECM/integrin interactions. Expression of uPA, uPAR, and PAI-1 was significantly enhanced when human ovarian cancer cells (OV-MZ-6) were cultivated on fibronectin or collagen type IV. In contrast, VN induced down-regulation of uPA and uPAR while increasing PAI-1 by up to 4-fold. VN-dependent decrease of uPA protein was paralleled by a significant reduction of uPA promoter activity that was even more pronounced upon alpha(v)beta(3) overexpression and depended on the presence of intact Rel protein-binding sites. The activity of Rel transcription factors was also significantly reduced upon alpha(v)beta(3)-mediated cell adhesion to VN. The activity of the Rel-unresponsive PAI-1 promoter was up to 5-fold induced as a function of alpha(v)beta(3)/VN interaction. Thus, the balance between available concentrations of uPA, uPAR, PAI-1, and integrins in human ovarian cancer cells might provide a switch within the regulation of their invasive phenotype.

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that beta(3)-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition beta(3)-integrin regulates u-PAR expression. Overexpression of beta(3)-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (-1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced beta(3)-integrin expression. A region between -398 and -197 bp of the u-PAR promoter was critical for beta(3)-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at -248 bp substantially impaired the ability of beta(3)-integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when beta(3)-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the beta(3)A-integrin isoform but not by other beta(3)-integrin isoforms and required the cytoplasmic membrane NITY(759) motif. Moreover, overexpression of the short but not the long isoform of the beta(3)-integrin adapter protein beta(3)-endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of beta(3)-integrin and u-PAR at the cell surface, beta(3) signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

Objective: Activated platelets induce alterations of chemotactic and adhesive properties of endothelial cells, a critical initial step in atherogenesis. We investigated the effect of transient interaction of activated platelets with cultured human umbilical vein endothelial cells (HUVECs) on secretion of monocyte chemoattractant protein-1 (MCP-1), a key molecule in monocyte chemotaxis and transmigration., Methods and Results: Transient interaction of alpha-thrombin-activated platelets with endothelial cells for 10-120 min substantially induced endothelial secretion of MCP-1, monocyte chemotaxis and adhesion to HUVECs. Platelet-induced secretion of MCP-1 and monocyte-endothelium adhesion was reduced by the MAP kinase p38-specific inhibitor SB203580, but not by other kinase inhibitors including PD98059, wortmannin, or rapamycin. In addition, activated platelets induced transcription of a luciferase reporter construct containing a MCP-1 promotor, an effect that could be inhibited by SB203580. Overexpression of dominant-negative mutants of MAP kinase p38, CSBP2-(D168A) and CSBP2-(T180E,Y182E) reduced platelet-induced expression of MCP-1., Conclusions: Activation of the p38 MAP kinase and consecutive endothelial secretion of MCP-1 induced through transient interaction of activated platelets might play an important role in atherogenesis.

PURPOSE: To demonstrate a conventional and a new therapeutic method of 3D treatment planning in maxilla tumors, the process of 3D treatment planning and its significance and to compare these two methods. METHOD: We performed 2D and 3D treatment plans. The ADAC planning system was used in the 3D treatment planning. CT and MRI scans were taken on the target volume and on each scan we demarcated the target volume and the critical organs. The irregular fields were obtained by 3D graphic reconstruction provided by the treatment planning programme. RESULTS: Compared to the conventional treatment planning more favourable dose distribution was obtained within the target volume and the radiation burden of the critical organs was kept under their tolerance doses. CONCLUSION: In conformal 3D treatment planning the shape and size of the irradiated volume are in good conformity with those of the target volume. In this way the radiation burden of the critical organs and adjacent intact tissues can be reduced.

Several cell biological studies have shown that the invasiveness of a variety of tumors depend on the regulated expression of proteolytic enzymes that degrade the surrounding extracellular matrix and dissociate cell-cell and/or cell-matrix attachments. One such enzyme, the serine protease urokinase-type plasminogen activator (uPA), converts enzymatically inactive plasminogen into the widely acting protease plasmin, which degrades several extracellular matrix components and also activates proenzyme forms of matrix metalloproteases. Thus, uPA is a central molecule in pericellular proteolysis (1-1). uPA (as well as other factors of the plasminogen activator system, the cell surface-associated uPA receptor [uPAR], and the plasminogen activator inhibitor type-1 [PAI-1]) is an important prognostic factor predicting relapse-free and/or survival in patients with a variety of solid malignant tumors including ovarian cancer; in all cases, high levels of uPA are associated with a poor prognosis (4-6).

Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth. Analogous to Ras, constitutively activating point mutations of Rac1 cause tumorigenic transformation of cell lines. However, there is no information about whether Rac1 is also mutated in vivo. After RT - PCR of Rac1, several clones of seven benign and 10 malignant breast cancer tissues as well as eight breast cancer cell lines were sequenced. Only single-nucleotide polymorphisms of Rac1 could be detected, and none of these corresponded to constitutively activating point mutations that have been used in cell lines for transformation. While sequencing Rac1 in breast tissues, a new Rac1 isoform with an insertion of 19 codons within the reading frame of Rac1 close to switch region II was identified and named Rac1b. The Rac1b protein acts like a fast cycling GTPase in GTP binding and hydrolysis assays. In Northern and Western blot experiments both Rac1 RNA and Rac1 protein had a significantly higher expression in breast cancer tissues compared to normal breast tissue samples. Immunohistochemical staining of Rac1 showed weak Rac1 expression in benign breast disease but high expression level in ductal carcinoma-in-situ, primary breast cancer, and lymph node metastases. In addition, breast tumor cells from patients with recurrent disease had Rac1 expression at the plasma membrane, suggesting activation of Rac1, in patients with aggressive breast cancer. Oncogene (2000).

Keratinocytes synthesize and secrete urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. When bound to urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator proteolytically converts surface bound plasminogen to plasmin, which in turn cleaves many extracellular components leading to pericellular proteolysis. The activation of the urokinase system has been observed during re-epithelialization of skin wounds and in lesions of the autoimmune blistering skin disease pemphigus. As pemphigus is photoinducible, we investigated the effect of ultraviolet B on urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor expression in the epidermal keratinocyte cell line A431. Ultraviolet B increased cellular and secreted urokinase-type plasminogen activator protein (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) 24 h postirradiation. Northern blot analysis indicated that ultraviolet B increased urokinase-type plasminogen activator receptor mRNA. Compared with a more rapid mRNA induction by epidermal growth factor (maximal after 4 h) the ultraviolet B response was maximal after 24 h and prolonged up to 36 h. The mRNA induction was not dependent on protein synthesis as judged by cycloheximide incubation. Ultraviolet B did not influence urokinase-type plasminogen activator receptor mRNA stability (actinomycin D incubation). A transiently transfected chloramphenicol acetyltransferase-reporter construct containing a -398/+51 urokinase-type plasminogen activator receptor promoter fragment was activated when cells were exposed to ultraviolet B. This induction was almost completely abolished by mutating a -182/-176 AP-1 binding sequence. Ultraviolet B increased the binding capacity at this AP-1 motif in electrophoretic mobility shift assays. These data identify a distinct transcriptional mechanism by which ultraviolet B induces urokinase-type plasminogen activator receptor. The epidermal induction of components of the proteolytic urokinase system by ultraviolet B may help explain the photoinducibility of pemphigus lesions.

Objective: This study was performed to determine the prognostic significance and the biological function of the structure protein keratin 18 (K18) in a group of patients with breast cancer., Material and Methods: Paraffin sections from the primary tumors in 80 patients with breast cancer were examined for the expression of K18 by immunohistochemical staining with the monoclonal antibody CK2. The intensity of the expression measured by an immune reactive score (IRS) was compared with clinicopathological variables and follow-up data up to 10 years. Additionally K18 expression in different human breast cancer cell lines with well defined metastatic behavior were investigated to confirm the clinical data., Results: A marked positive staining was observed in 13 (16.2%) women. Mortality rate in this group was 7.7% and 46.3% in the group with a low K18 expression. The recurrence rate was with 15.4% significantly lower in the positive group than in the negative group with 56.7%. Irrespective of the morphological tumor stage, almost all patients with strong K18 expression were still free of disease after 10 years. This clinical finding is supported by observations in cell cultures., Conclusions: In our collective K18 expression was found to be an independent and significant predictor for disease-free and overall survival.

Invasive growth and formation of metastases involve complex interactions between tumour cells, host cells and components of the extracellular matrix. Retinoids, a group of vitamin A derivatives, modulate cell growth and differentiation and have been found to suppress tumour cell invasion in vitro and formation of metastases in vivo. The aim of our study was to investigate changes in proliferation and invasion through membrane barriers in vitro of seven human melanoma cell lines, established from human primary melanomas or metastases, in response to treatment with retinoic acid (RA). These changes were compared with the expression regulation of molecules that have been identified as targets of RA-mediated signal pathways. Invasiveness in vitro was correlated with the origin of the cell lines and was significantly higher in the lines derived from metastases. In all the cell lines proliferation and chemotaxis were inhibited by 10(-5) M RA, but the cell lines established from metastases were significantly more sensitive with respect to inhibition of invasion by RA. The specific expression patterns of MMP-1 and TIMP-2 were detected and regulated by RA in almost all cell lines, whereas expression of MMP-2 and TIMP-1 was not influenced by RA treatment. The most striking difference between the cell lines was a strong downregulation of transforming growth factor-beta (TGF-beta) expression in cell lines derived from metastases when treated with RA in contrast to cell lines from primary melanomas. These data provide evidence that RA modulates growth, chemotaxis and invasion in a broad panel of melanoma cell lines derived both from primary non-metastasized melanomas and metastases. However, distinct molecular mechanisms are involved in mediating these effects.

The long-term cause-specific survival results of papillary thyroid cancer patients in Hungary are (78%) worse than the best international data (90%). The authors have recited its causes through the case of a young papillary thyroid cancer patient during the diagnostics (aspiration cytology, diagnostic imaging technics, DNA-analysis) the treatment (surgery-external radiotherapy-radioiodine treatment) and the follow-up (HTG, diagnostic imaging).

The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix degradation in part by accelerating plasmin formation at the cell surface. We previously reported that u-PAR expression is elevated in colon cancer cell lines characterized by their in vitro invasive capacity. Since, u-PAR expression is increased by a variety of growth factors, which signal through the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), we determined if these mitogen-activated protein kinases (MAPKs) regulate u-PAR expression in two cultured colon cancer cell lines. An in-gel kinase assay showed that ERK1 activity was considerably higher in RKO cells, which display > or = 10(5) receptors/cell, than the GEO cells which have approximately 10(4) urokinase receptors per cell. The expression of either an ERK-inactivating phosphatase (CL100), or a kinase-defective ERK1, decreased the activity of a u-PAR promoter-driven CAT reporter in RKO cells. Immune complex kinase assays indicated that the constitutive ERK1 activity in RKO cells was largely a result of an activated MEK1. Further, treatment of RKO cells with a specific inhibitor (PD 098059) of MEK1 activation, which diminished ERK1 activity, reduced the amount of urokinase specifically bound to the cell surface and this was associated with reduced laminin degradation. The expression of a dominant negative c-Raf-1 also reduced u-PAR promoter activity suggesting that MEK1 activation involved an activator at, or upstream, of this serine-threonine kinase. Transfection of the u-PAR-deficient GEO cells with a constitutively activated MEK1 expression construct up-regulated u-PAR promoter activity. Similarly treatment of GEO cells with a phosphatase inhibitor (sodium vanadate) caused a dose-dependent increase in ERK1 activity which paralleled increased cell surface binding of urokinase. Taken together, these data suggest that elevated u-PAR expression, in at least a sub-population of colon cancer, is partly a consequence of a constitutively activated ERK-1-dependent signaling cascade.

The 92 kDa type IV collagenase (MMP-9), which degrades type IV collagen, has been implicated in tissue remodeling. The purpose of the current study was to determine the role of Jun amino-terminal kinase (JNK)- and extracellular signal-regulated kinase- (ERK)-dependent signaling cascades in the regulation of MMP-9 expression. Towards this end, we first determined the transcriptional requirements for MMP-9 promoter activity in a cell line (UM-SCC-1) which is an avid secretor of this collagenase. Transfection of these cells with a CAT reporter driven by progressive 5' deleted fragments of the MMP-9 promoter indicated the requirement of a region spanning -144 to -73 for optimal promoter activity. DNase I footprinting revealed a protected region of the promoter spanning nucleotides -91 to -68 and containing a consensus AP-1 motif at -79. Mutation of this AP-1 motif practically abolished the activity of the MMP-9 promoter-driven CAT reporter. Mobility shift assays indicated c-Fos and Jun-D bound to this motif and transfection of the cells with a mutated c-Jun, which quenches the function of endogenous Jun and Fos proteins, decreased MMP-9 promoter activity by 80%. UM-SCC-1 cells contained a constitutively activated JNK and the expression of a kinase-deficient JNK1 reduced the activity of a CAT reporter driven either by the MMP-9 promoter or by three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter. Conditioned medium collected from UM-SCC-1 cells transfected with the dominant negative JNK1 expression vector diminished 92 kDa gelatinolysis. Similarly, interfering with MEKK, which lies upstream of JNK1, using a dominant negative expression vector reduced MMP-9 promoter activity over the same concentration range which repressed the AP-1-thymidine kinase CAT reporter construct. UM-SCC-1 cells also contained a constitutively activated ERK1. MMP-9 expression, as determined by CAT assays and by zymography, was reduced by the co-expression of a kinase-deficient ERK1. Interfering with MEK1, which is an upstream activator of ERK1, either with PD 098059, which prevents the activation of MEK1, or with a dominant negative expression construct, reduced 92 kDa gelatinolysis and MMP-9 promoter activity respectively. c-Raf-1 is an upstream activator of MEK1 and a kinase-deficient c-Raf-1 expression construct decreased the activity of a promoter driven by either the MMP-9 promoter or three tandem AP-1 repeats. Conversely, treatment of UM-SCC-1 cells with PMA, which activates c-Raf-1, increased 92 kDa gelatinolysis. These data suggest that MMP-9 expression in UM-SCC-1 cells, is regulated by JNK- and ERK-dependent signaling pathways.

This study was performed to determine if keratin 18 (K18) has prognostic significance in breast cancer cell lines and patients with breast carcinoma. Paraffin sections of primary breast carcinoma tumors and human breast carcinoma cell lines were examined for K18 expression by immunohistochemical staining with the monoclonal antibody CK2. K18 protein expression was low in highly metastatic cell lines and, conversely, high in weakly metastatic cell lines, suggesting that it may function as a prognostic indicator. K18 expression was consequently examined in 134 patients with breast cancer. The staining intensity was compared with clinicopathological variables and follow-up data spanning 8 years. A definitive positive staining was observed in 22 (16.4%) women. The mortality rate was 4. 5% in the K18-positive group and 44.6% in the K18-negative group. Multivariate analysis found K18 expression to be an independent and significant predictor for overall survival.

The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix proteolysis by accelerating plasmin formation at the cell surface. The present study was undertaken to identify elements in the u-PAR promoter required for the elevated expression of this binding site. Toward this end, we used two cultured colon cancer cell lines; one (RKO) has a transcriptionally activated u-PAR gene, and the other (GEO) overexpresses the receptor only after phorbol ester treatment. A chloramphenicol acetyltransferase (CAT) reporter driven by 398 nucleotides of 5' regulatory sequence of the u-PAR gene was strongly activated in the RKO cells, which displays approximately 3 x 10(5) receptors/cell. A region of this promoter between -197 and -8 was required for optimal expression, as indicated using a CAT reporter driven by 5' deleted fragments. DNase I footprinting revealed three protected regions (I, -190 to -171; II, -148 to -124; and III, -99 to -70) in this part of the promoter. Mutation of an AP-1 binding site at -184 within region I reduced activation of the promoter by 85%. Deletion of either region II or III also reduced promoter activity by over 60%. An oligonucleotide spanning the AP-1 motif at -184 bound, specifically, nuclear factors from RKO cells, and antibodies specific for Jun-D, c-Jun, or Fra-1 proteins supershifted the complex indicating the presence of these proteins. The amount of these factors was reduced in GEO cells in which the u-PAR gene is only weakly transcriptionally activated. Expression of a vector encoding a wild-type Jun-D cDNA increased u-PAR promoter activity in GEO cells. Conversely, transfection of RKO cells with a transactivation domain-lacking Jun-D expression construct resulted in a dose-dependent decrease in u-PAR promoter activity. Treatment of GEO cells with phorbol ester increased u-PAR mRNA and the activity of a CAT reporter driven by the wild-type but not the AP-1 (-184)-mutated u-PAR promoter, and this was associated with a strong induction in the amount of Jun-D, c-Jun, and c-Fos. Methylation interference studies using a fragment of the u-PAR promoter (spanning -201 to -150) bound with nuclear extracted proteins from RKO cells, and phorbol 12-myristate 13-acetate-treated and -untreated GEO cells showed that the contact points corresponded to the AP-1 binding site at -184. Thus, the elevated expression of u-PAR in RKO cells, which constitutively produces this binding site, as well as in phorbol 12-myristate 13-acetate-stimulated GEO cells requires an AP-1 motif located 184 bp upstream of the transcriptional start site.

The urokinase-type plasminogen activator contributes to tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. We undertook a study to determine the role of the extracellular signal-regulated kinases (ERKs) in the regulation of urokinase-type plasminogen activator expression in a squamous cell carcinoma cell line (UM-SCC-1) that contains a transcriptionally activated urokinase-type plasminogen activator gene. Transient transfection studies using a CAT reporter driven by the urokinase-type plasminogen activator promoter, which had progressive 5' deletions or which had been point-mutated, indicated the requirement of binding sites for AP-1 (-1967) and PEA3 (-1973) for its maximal activation. Expression of a mutant jun protein, which lacks the transactivation domain, caused a dose-dependent repression of a CAT reporter driven by either the urokinase-type plasminogen activator promoter or three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter indicating the importance of AP-1-binding transcription factor(s) in the regulation of urokinase-type plasminogen activator synthesis. Mobility shift assays with UM-SCC-1 nuclear extract revealed binding of fos and junD proteins to an oligonucleotide spanning the AP-1 site at -1967. In-gel kinase assays indicated the constitutive activation of ERK1, which regulates fos synthesis via phosphorylation of p62TCF, but not ERK2, in UM-SCC-1 cells. Moreover, the expression of a dominant-negative ERK1, but not ERK2, repressed urokinase-type plasminogen activator promoter activity. Similarly, interfering with the function of the c-raf serine-threonine kinase, which lies upstream of ERK1, by the expression of a kinase-inactive c-raf repressed the activity of a CAT reporter driven by either the urokinase-type plasminogen activator promotor or tandem AP-1 repeats. These data suggest that urokinase-type plasminogen activator expression in UM-SCC-1 cells is regulated partly by an ERK1, but not ERK2, -dependent signaling pathway.

The urokinase-type plasminogen activator plays a central role in tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. Urokinase expression is transcriptionally regulated by a variety of cytokines including TNF-alpha. The present study was undertaken to identify key transcription factor binding sites in the urokinase promoter necessary for the TNF-alpha-dependent induction of urokinase expression. TNF-alpha treatment of a squamous cell carcinoma cell line, UM-SCC-1, which produces no detectable TNF-alpha, led to a dose-dependent increase in urokinase secretion, thus reflecting a more abundant mRNA. Transient transfections of UM-SCC-1 cells with a CAT reporter driven by 5' deletion fragments of the urokinase promoter indicated that a sequence spanning -2109 to -1870, which contained binding sites for AP-1 and PEA3 was required for the stimulation by TNF-alpha. Mutation of an AP-1 binding site at -1967 and a PEA3 motif at -1973 completely abrogated the inductive effect of TNF-alpha on urokinase promoter activity. Mobility shift assays indicated the presence of a jun-containing factor(s) which bound specifically to the AP-1 sequence present in the urokinase promoter. The amount and/or activity of this factor(s) was greatly enhanced by TNF-alpha treatment. UM-SCC-1 cells transiently transfected with a CAT reporter driven by 3 tandem AP-1 binding sites demonstrated increased CAT activity following TNF-alpha treatment. Thus, the induction of urokinase expression by TNF-alpha is likely to involve the altered expression and/or activity of transcription factors which bind to the AP-1 and PEA3 target sequences in the urokinase promoter.

The HER2/neu (c-erbB2) protooncogene, which encodes a transmembrane receptor (p185neu), contributes to tumor cell invasion/metastasis through mechanism(s) which are, at present, poorly defined. Since basement membrane degradation is a prerequisite for tumor progression, we undertook a study to determine if the expression of urokinase, a key protease implicated in extracellular matrix proteolysis, was regulated by this oncogene. Stable overexpression of a cDNA encoding HER2/neu in H460 lung cancer cells led to elevated secretion of urokinase which was a consequence of a higher level of protease mRNA. Transfection of the HER2/neu-overexpressing B 104-1 cells with a CAT reporter construct driven by the urokinase promoter, gave rise to increased CAT activity when compared with parental NIH3T3 cells, which have low levels of HER2/neu, suggesting that the protooncogene can enhance urokinase promoter activity. Since the enhanced expression of HER2/neu results in increased tumor invasion/metastasis (1), these data suggest that, at least in vitro, HER2/neu-induced expression of urokinase may contribute to tumor progression in p185neu-positive cancers.

A previous investigation (Matsumoto et al., J. Oral Pathol. Med., 18: 498-501, 1989) has shown that the in vitro invasion of a collagen gel by squamous cell carcinoma can be substantially augmented in the presence of fibroblasts. Therefore, we undertook a study to determine if the production of collagenase(s) by a squamous cell carcinoma cell line, UM-SCC-1, was up-regulated by fibroblasts. Cocultivation of UM-SCC-1 cells with MDA-TU-138 fibroblasts, both established from the oral cavity, resulted in a dose-dependent increase in the activity of a M(r) 92,000 gelatinase as shown by zymography. Augmented M(r) 92,000 gelatinase activity was a consequence of the stimulation of the UM-SCC-1 cells by a soluble, fibroblast-derived factor since this effect could be reproduced with fibroblast-conditioned medium but not with glutaraldehyde-fixed fibroblasts. The increased M(r) 92,000 gelatinolytic activity could be accounted for by an increase in M(r) 92,000 type IV collagenase (MMP-9) protein, as demonstrated by Western blotting for this metalloproteinase. Trypsin treatment of the fibroblast-conditioned medium abolished its ability to increase MMP-9 secretion by UM-SCC-1 cells. Furthermore, fractionation of the fibroblast-conditioned medium revealed a M(r) 3,000-10,000 soluble factor(s) which was responsible for the augmented production of MMP-9 by UM-SCC-1 cells. To determine if the increased production of MMP-9, in response to the fibroblasts, was a consequence of increased promoter activity, UM-SCC-1 cells were transiently transfected with a chloramphenicol acetyltransferase reporter driven by the MMP-9 promoter and plated on plastic or on a monolayer of MDA-TU-138 fibroblasts. A 4-5-fold stimulation of MMP-9 promoter activity was observed with UM-SCC-1 cells plated with the MDA-TU-138 fibroblasts, when compared with similarly transfected cells recultured on plastic. In conclusion, we have demonstrated that MMP-9 expression in a squamous cell carcinoma cell line is augmented by a fibroblast-derived protein(s). This finding indicates a role for stromal cells in the regulation of MMP-9 expression in squamous cell carcinoma. The ability of fibroblasts to regulate MMP-9 expression in tumor cells in vitro may explain the observation that the amount of M(r) 92,000 type IV collagenase mRNA in tumor cells is highest at the tumor:stromal interface of resected squamous cell carcinoma.

The clinical and laboratory data from a cross-sectional and a longitudinal study of healthy individuals between 50-89 years of age and that of centenarians were compared. The examination showed that in healthy elderly subjects and centenarians most of the clinical laboratory findings were in the normal range. However, age-related changes could be observed in erythrocyte sedimentation rate, in serum albumin level, and in the frequency of rheuma factor. Gender difference could be seen in the HDL-cholesterol level. Age and sex differences were observed in the hematocrit-, and hemoglobin-values, in serum creatinine-, total cholesterol-, serum IgG, IgA, IgM-levels, and in the frequency of the occurrence of antinuclear factor. The examinations demonstrated that lower hemoglobin and hematocrit values, elevated total-cholesterol level, the presence of antinuclear and rheuma factor, and elevated systolic blood pressure were more frequent in females than in males. It could be stated, however, that the mentioned changes did not prevent females from having a longer life span than males.

The effects of lymph stasis on the histological and biochemical properties of the coronary arterial wall and on the coronary circulation were studied in 72 dogs. Cardiac lymph stasis was produced in 52 dogs by cardiac lymphatic blockade whereas in 20 dogs only a sham operation was performed. Blockade of cardiac lymph drainage promoted characteristic injury to the coronary arteries including subendothelial edema with plasma inbibition, interstitial and intracellular edema in the tunica media with degeneration in the smooth muscle layer, swelling of the adventitial space with dilated lymph vessels and, later, fibrosis. The biochemical properties of the coronary arterial wall also were adversely affected by cardiac lymph stasis. Thus, the collagen and hexosamine content of the coronary arteries increased and the metabolism of the coronary wall shifted in an anaerobic direction. Whereas coronary blood flow was slightly decreased with lymph blockade, the coronary circulatory reserve capacity and the adaptability of the coronary vascular system was markedly reduced. The histological changes were most apparent in the smaller coronary arteries. The coronary microvasculature was also pathologically altered with the development of numerous coronary arteriovenous microshunts. These findings in conjunction with other experimental and clinical information suggest that impaired cardiac lymph drainage contributes to the pathogenesis and progression of coronary artery disease.

Objective: Analyzing the expression of keratins has proved to be valuable for identifying of pathways of epithelial differentiation. In stratified epithelia K10 and K13 are representative for either the keratinizing (epidermal-type) or the nonkeratinizing pathway., Study Design: We have investigated keratin expression in "normal" vaginal epithelium from 30 women, applying two-color immunofluorescence with monoclonal antibodies to K10 and K13 on cryostat sections and cell smears., Results: A differential expression pattern of vaginal cells dependent on their localization within the epithelium was found. In cells of the first suprabasal layers differentiation began and became identifiable by a weak expression of K13. The adjacent layers displayed cells that concurrently expressed K10 and K13. In contrast, cells within the superficial strata expressed exclusively either one of the two keratins., Conclusion: Thus vaginal epithelium appears to be mosaic in differentiation, showing simultaneous expression of keratins K10 and K13, thought to be representative for distinct routes of differentiation.

The present work reports on a 20-year longitudinal study in Budapest. Eight-hundred-sixty-four subjects who considered themselves healthy, took part in the clinical examination that included women age 55 years and older, and men of 60 years older at the start of the study. The data of the 3080 clinical check-ups have been processed and analyzed. Diseases of the circulatory system occurred most frequently. Ischemic heart disease, cerebrovascular disease, and pulmonary emphysema showed a definite age-dependency. Cataract, fundus sclerosis, decline of visual acuity, as well as hyperplasia of prostate also proved to be age-dependent. The occurrence of osteoporosis and spondylarthrosis increased significantly with age. In the mentioned diseases, correlations were found between the frequency and the number of follow-up years. In relation to our study, we can state that the importance of the risk factors decrease with aging. The pathological effects of the risk factors occur in earlier in life and the truly endangered persons would not grow old.

Physiological age-related changes were investigated by cross-sectional and longitudinal studies. The data of the study were derived from volunteers admitted to the Gerontology Center in Budapest. In the follow-up study 864 subjects took part, and in the cross-sectional study 2,281 individuals. The 20-year longitudinal study and the cross-sectional examination showed that from a clinical standpoint the most important observation is that several biochemical data do not change with age. No changes were found in the values in erythrocyte sedimentation, hemoglobin, hematocrit, serum total protein, albumin, liver function, glucose, creatinine, triglyceride, and HDL-cholesterol levels. Changes were observed in the levels of HDL-cholesterol, immunoglobulins, rheumatoid factors, antinuclear factors, morphology of lymphocytes, serum TSH and T3 concentration, and systolic blood pressures.

The effect of lymph stasis on the histological, biochemical, and elastic properties of the femoral artery were studied after regional lymphatic blockade in 36 dogs. Dogs were sacrificed 4-21 days after operation. Histologic changes of the femoral arterial wall (interstitial edema, degeneration in the muscle layer or media, thickened adventitia with dilated lymph vessels, and fibrosis) developed after regional lymphatic blockade. Characteristic metabolic alterations of the arterial wall (anaerobic catabolism of carbohydrate, increased lactate and glycosamine content) accompanied the morphological changes. Distensibility of the femoral artery decreased and greater elastic stiffness developed after regional lymphatic blockade. These results in conjunction with other experimental and clinical data support the concept that insufficient lymphatic transport within the blood vessel wall may contribute to the genesis and progression of arteriopathies.

To investigate the influence of smoking on the aging immune system, the following parameters were determined in elderly smokers and non-smokers: presence of emphysema; absolute number of leucocytes, lymphocytes, T cells and B cells bearing membrane-bound IgG; serum IgG, IgA, IgM levels, occurrence of rheumatoid and antinuclear factors, and natural antibody level (against different bacteria). Ultrastructural disorders in lymphocytes were also investigated. The number of leucocytes, the levels of serum IgA, and the prevalence of autoantibodies were higher, and the natural antibody level was lower in smokers than in non-smokers. Giant mitochondria and cytoplasmic disorders were found more frequently in smokers than in non-smokers, and in an even higher rate in smokers with emphysema. These findings suggest that smoking may accelerate the appearance of age-dependent (especially disease-related) immunological changes.