Formation Of Benign Tumors Research Articles

NF1 mice: smaller brains but no tumours

Neurofibromatosis type 1 is a common neurological disease with a worldwide incidence of 1 in 3500. The major clinical features of this disease include formation of benign tumours along peripheral and optic nerves (neurofibromas and gliomas), abnormal distribution of melanocytes (café-au-lait spots) and, in some cases, learning disabilities. Neurofibromatosis type 1 has genetic aetiology with a dominant pattern of inheritance and is caused by mutations in the tumour suppressor gene neurofibromin (NF1). This gene appears to be involved in inactivation of the Ras signalling pathway and hence negatively regulates cell proliferation. However, the role of NF1 in development and cognition is unclear.

Platelet-derived growth factor mediated altered expression and regulation of ornithine decarboxylase in H-ras-transformed cell lines

This study demonstrates a novel link between alterations in platelet-derived growth factor (PDGF) regulation of ornithine decarboxylase (ODC) expression during malignant conversion. H-ras-transformed cell lines exhibited PDGF-mediated alterations in ODC gene expression. These alterations involved transcriptional, posttranscriptional, and cycloheximide-mediated events. PDGF-mediated alterations in ODC gene expression in NR3 cells (capable of only benign tumour formation) were ras-dependent, involved a tyrosine kinase activity and mitogen-activated protein (MAP) kinase-mediated signalling events, and were independent of both protein kinase C (PKC) events and pertussis toxin-sensitive (PTS) G-protein-mediated signalling. PDGF-mediated alterations in ODC gene expression in C2 cells [capable of malignant progression (metastasis formation)] were ras-dependent, required a tyrosine kinase activity, involved both MAP kinase-mediated events and phosphatidylinositol-3-kinase (PI-3-kinase)-mediated events, and were dependent upon PTS G-protein-mediated signalling but independent of PKC-mediated events. PDGF-mediated regulation of ODC gene expression changes in response to H-ras-mediated cellular transformation and malignant progression.

Phorbol ester tumour promoter mediated altered expression and regulation of matrix metalloproteinase-2 in a H-ras transformed cell line capable of benign tumour formation.

The matrix metalloproteinases (MMPs) are thought to play key roles in tumour formation and malignant progression. The present study demonstrates alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in response to the phorbol ester tumour promoter, PMA, in a H-ras transformed cell line, NR3, which is capable of benign tumour formation. PMA treatment of NR3 cells resulted in decreased expression of MMP-2 mRNA levels. Following a lag period, an accompanying change in gelatinolytic activity was also found. These PMA-mediated alterations in MMP-2 mRNA levels were independent of de novo protein synthesis and involved both transcriptional and post-transcriptional events. Most notably, PMA regulates MMP-2 mRNA expression through a mechanism involving message de-stabilization. Additionally, protein kinase C mediated events were found to play a role(s) in the regulation of MMP-2 message expression in NR3 cells. This study demonstrates several novel aspects regarding the regulation of MMP-2 expression in a H-ras transformed cell line and thereby provides further insight into the altered growth regulatory programs associated with H-ras mediated cellular transformation.

S-Adenosylmethionine decarboxylase gene expression is regulated by the cAMP signal transduction pathway in H-ras transformed fibrosarcoma cells capable of malignant progression.

The hypothesis that H-ras transformed cells contain alterations in signalling pathways important in controlling the expression of S-adenosylmethionine decarboxylase, (SAMDC) a highly regulated activity in the biosynthesis of polyamines was tested. Mouse 10 T1/2 fibroblasts and H-ras transformed cell lines of varying degrees of malignant potential were treated with agents which affect cAMP levels within cells. Elevations in SAMDC expression were noted in H-ras transformed metastatic C3 cells, which were not observed in either parental, non-transformed 10 T1/2 fibroblast cells, or in ras transformed NR3 cells, which are only capable of benign tumour formation. Forskolin, a stimulator of cAMP synthesis, was able to increase SAMDC enzyme activity but the response which occurred was dependent upon the cellular phenotype expressed. Actinomycin D pre-treatment of C3 cells prior to exposure to forskolin did not abrogate the elevation observed in SAMDC gene expression suggesting that this was not a transcriptional process mediated event. Forskolin pre-treatment of C3 cells did result in a marked increase in the half-life of SAMDC mRNA transcripts suggesting a role for post-transcriptional stabilization. Furthermore, cycloheximide treatment of malignant C3 cells resulted in elevated SAMDC mRNA levels. Treatment of malignant C3 cells with both cycloheximide and forskolin together resulted in a further additive elevation in SAMDC message levels. Cycloheximide treatment alone was found to affect the half-life of SAMDC mRNA through a mechanism of post-transcriptional stabilization. Additionally, altered SAMDC gene expression in C3 cells which occurred in response to cAMP alterations, was enhanced by stimulation of a protein kinase C pathway suggesting possible interactions between protein kinase C-and cAMP-mediated pathways which affect the regulation of SAMDC expression in highly malignant C3 cells. These results demonstrate aberrant regulation of signalling pathways involved in controlling SAMDC gene expression in H-ras transformed cells capable of malignant progression and provide further insight into the altered growth regulatory program associated with H-ras mediated cellular transformation and malignant progression. J. Cell. Biochem. Suppl. 36: 209-221, 2001.

Insulin like growth factor-1 selectively regulates the expression of matrix metalloproteinase-2 in malignant H-ras transformed cells.

The present study demonstrates alterations in the regulation of matrix metalloproteinase-2 (MMP-2) expression in response to insulin like growth factor-1 (IGF-1) in a H-ras transformed cell line, C3, which is capable of metastasis formation. These changes in MMP-2 expression in response to IGF-1 treatment did not occur in either non-transformed parental 10 T 1/2 cells or in H-ras transformed cells (NR3 cells) which are capable of benign tumour formation. IGF-1 treatment of C3 cells resulted in increased expression of MMP-2 gelatinolytic activity and increased expression of MMP-2 mRNA levels. The IGF-1 mediated alterations in MMP-2 mRNA levels were dependent upon de novo protein synthesis and independent of transcriptional events, but dependent upon post-transcriptional regulatory events. Most notably, IGF-1 can regulate MMP-2 mRNA expression in C3 cells through a mechanism involving MMP-2 message stabilization. This study demonstrates aspects of the temporal regulatory mechanisms of MMP-2 expression in response to insulin-like growth factor-1 in a H-ras transformed fibrosarcoma cell line capable of metastasis formation and thereby, provides further insight into the altered growth regulatory program associated with H-ras mediated cellular transformation and malignant progression.

Tumour promoter mediated altered expression and regulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase in H-ras-transformed fibrosarcoma cell lines

Alterations in cellular growth are important in the progression of malignant disease. Cell growth regulation by tumour promoters can be complex. The present study demonstrates a novel link between alterations in phorbol ester tumour promoter mediated regulation during malignant conversion and the expression of ornithine decarboxylase and S-adenosylmethionine decarboxylase, key rate-limiting and regulatory activities in the biosynthesis of polyamines. H-ras-transformed mouse 10 T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible phorbol ester tumour promoter mediated changes in ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) gene expression. Selective induction of ODC and SAMDC gene expression was observed, since in contrast to nontransformed parental 10 T1/2 cells, ras-transformed cells capable of benign tumour formation (NR3 cells) and ras-transformed cells capable of metastasis formation (C2 cells) exhibited marked alterations in the levels of ODC and SAMDC gene expression. Increased ODC gene and SAMDC gene expression in response to phorbol-12-myristate-13-acetate (PMA) treatment was found to involve transcriptional events in both NR3 cells and in C2 cells. Post-transcriptional events also played a role in the regulation of ODC gene expression in NR3 cells and in C2 cells, and in the regulation of SAMDC gene expression in C2 cells but not in NR3 cells. In NR3 cells, alterations in ODC and in SAMDC gene expression was an event requiring de novo protein synthesis, whereas in highly malignant C2 cells, protein synthesis inhibition following cycloheximide treatment in cooperation with PMA resulted in an augmentation of both ODC and SAMDC gene expression. Evidence is presented to suggest that the PMA-mediated alterations in ODC and in SAMDC gene expression in NR3 cells and in C2 cells involved protein kinase C - mediated events. The status of the cellular polyamine levels was also an important determinant of the PMA-mediated alterations that occurred in ODC and in SAMDC expression in these H-ras transformed cells. Collectively, these results suggest that PMA can modulate ODC and SAMDC expression in H-ras transformed cells and that the mechanisms involved in the PMA- mediated regulation of ODC and SAMDC gene expression changes as a function of H-ras mediated cellular transformation and malignant progression. This study further suggests a mechanism of PMA stimulation of transformed cells wherein early alterations in the regulatory control of ODC and SAMDC gene expression are important and critical.

Insulin-mediated alterations in S-adenosylmethionine decarboxylase expression in H-ras transformed cells of varying degrees of malignancy

Cell growth regulation is a highly complex process. The present study demonstrates a novel link between alterations in insulin-mediated regulation during malignant conversion and the expression of S-adenosylmethionine decarboxylase, a key regulatory activity in the biosynthesis of polyamines. H-ras transformed mouse 10 T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible insulin-mediated changes in S-adenosylmethionine decarboxylase gene expression . Selective induction of S-adenosylmethionine decarboxylase gene expression was observed, because, in contrast to nontransformed 10T 1/2 cells, only H-ras transformed cells capable of only benign tumour formation or H-ras transformed metastatic cells exhibited marked elevations in S-adenosylmethionine decarboxylase mRNA levels. Evidence for regulation of S-adenosylmethionine decarboxylase gene expression at both transcriptional and post-transcriptional levels was found . Evidence was also found for a cycloheximide sensitive regulator of S-adenosylmethionine decarboxylase gene expression in H-ras transformed metastatic cells, whose effect, in combination with insulin, resulted in a further augmentation of S-adenosylmethionine decarboxylase gene expression. This regulation was not present in H-ras transformed cells capable of only benign tumour formation. These results suggest that insulin can modulate S-adenosylmethionine decarboxylase gene expression in H-ras transformed cells and further suggests a mechanism of insulin stimulation of transformed cells wherein alterations in the regulatory activity of S-adenosylmethionine decarboxylase gene expression are critical and constitutes a part of an altered growth regulatory program associated with cellular transformation.

Genetic and cellular defects contributing to benign tumor formation in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a common inherited cancer predisposition syndrome. The NF1 gene product, neurofibromin, is hypothesized to function as a tumor suppressor and nearly all NF1 patients develop benign peripheral nerve tumors. These neurofibromas presumably arise from NF1 inactivation in S100(+)Schwann cells, but there is no formal proof for this mechanism. We demonstrate that fibro-blasts isolated from neurofibromas carried at least one normal NF1 allele and expressed both NF1 mRNA and protein, whereas the S100(+)cells typically lacked the NF1 transcript. Our findings further indicate that additional molecular events aside from NF1 inactivation in Schwann cells and/or other neural crest derivatives contribute to neurofibroma formation.

Human Papillomavirus Types 16 E6 and E7 Contribute Differently to Carcinogenesis

High-risk human papillomaviruses (HPVs) are etiologically implicated in human cervical cancer. Two viral genes, E6 and E7, are commonly found expressed in these cancer cells. We have previously shown that mice transgenic for the HPV-16 E6 gene or E7 gene, in which the E6 or E7 was expressed in the basal layer of epithelia, developed skin tumors. The spectrum of tumors derived from E6 and E7 mice differed, however; although most tumors derived from the E7-transgenic mice were benign, the majority of the tumors from the E6-transgenic mice were malignant. These findings led us to hypothesize that E6 and E7 play different roles in carcinogenesis. To assess at what stages in carcinogenesis E6 and E7 act, we treated the skin of K14E6- and K14E7-transgenic mice with chemical carcinogens known to contribute to distinct stages in carcinogenesis. Both E6 and E7 were found to synergize with chemical carcinogens in causing tumor formation. E6 was found to act weakly at the promotion stage of carcinogenesis in the formation of benign tumors but strongly at the progression stage which involves the malignant conversion of benign tumors. In contrast, E7 primarily affected the promotion stage of carcinogenesis. These results provide direct evidence that E6 and E7 contribute differently to carcinogenesis; E7 promotes the formation of benign tumors, and E6 acts primarily to accelerate progression of these benign tumors to the malignant stage. Consistent with this model, we found E6 and E7 to cooperate in inducing tumor formation in mice expressing both oncogenes.

P21(WAF1/Cip1) functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential.

p21(WAF1/Cip1) is one of the best characterized downstream targets of p53, and the growth suppressing function of this cyclin-dependent kinase inhibitor is well established. However, whether p21 exerts a tumor-suppressing function of its own remains to be established. We report here that, similarly to loss of p53, disruption of the p21(WAF1/Cip1) gene results in a markedly increased susceptibility to chemically induced skin carcinoma formation, whereas the number of papillomas is reduced. Previous evidence indicates that malignant versus benign keratinocyte tumor formation is likely to involve distinct target-cell populations with a different commitment to differentiation. In parallel with the increased susceptibility to carcinoma formation, loss of p21(WAF1/Cip1) was found to promote keratinocyte subpopulations with increased growth/differentiation potential, including clonal growth capability, reversible commitment to differentiation, and capability to generate all types of terminally differentiated keratinocytes present in vivo, not only in the interfollicular epidermis but also in hair follicles. Thus, these findings have revealed a function of p21 as a suppressor of malignant but not benign skin-tumor formation and a determinant of the growth/differentiation potential of keratinocyte subpopulations.

The human papillomavirus type 16 E6 gene alone is sufficient to induce carcinomas in transgenic animals.

High-risk human papillomaviruses (HPVs) are the causative agents of certain human cancers. HPV type 16 (HPV16) is the papillomavirus most frequently associated with cervical cancer in women. The E6 and E7 genes of HPV are expressed in cells derived from these cancers and can transform cells in tissue culture. Animal experiments have demonstrated that E6 and E7 together cause tumors. We showed previously that E6 and E7 together or E7 alone could induce skin tumors in mice when these genes were expressed in the basal epithelia of the skin. In this study, we investigated the role that the E6 gene plays in carcinogenesis. We generated K14E6 transgenic mice, in which the HPV16 E6 gene was directed in its expression by the human keratin 14 promoter (hK14) to the basal layer of the epidermis. We found that E6 induced cellular hyperproliferation and epidermal hyperplasia and caused skin tumors in adult mice. Interestingly, the tumors derived from E6 were mostly malignant, as opposed to the tumors from E7 mice, which were mostly benign. This result leads us to hypothesize that E6 may contribute differently than E7 to HPV-associated carcinogenesis; whereas E7 primarily contributes to the early stages of carcinogenesis that lead to the formation of benign tumors, E6 primarily contributes to the late stages of carcinogenesis that lead to malignancy.

Genetic basis of uterine leiomyoma: involvement of high mobility group protein genes

Uterine leiomyoma is a common benign smooth muscle tumor occurring in 20 to 30% of women over 30 years of age. Cytogenetic analysis of such tumors has revealed that over 60% of them have a normal karyotype but also that they sometimes carry recurrent chromosome aberrations, e.g., aberrations involving the short arm of chromosome 6 or the long arm of chromosome 12. Precise mapping of chromosome breakpoints of such recurrent chromosome translocations and molecular characterization of DNA regions immediately flanking such breakpoints have proven to be a successful approach to identify and isolate genes involved in tumor development. Recently, application of such a research strategy on translocations in uterine leiomyoma involving either chromosome 12q13-15 or 6p21 has led to the discovery that two members of the high-mobility group (HMG) protein gene family, HMGIC and HMGIY, are frequently rearranged in such tumors. The developmentally regulated HMGIC and HMGIY genes encode closely related, low-molecular-mass proteins, which are assumed to function as architectural factors in the nuclear scaffold and to be critical in the assembly of stereospecific transcriptional complexes. The frequent rearrangement of the HMGIC and HMGIY genes in uterine leiomyomas suggests that these genes are directly involved in the aberrant growth control observed in these tumors. Studies of a number of other benign solid tumors – most of them in tissues of mesenchymal origin – indicated that involvement of HMGIC is not restricted to uterine leiomyomas, suggesting that benign solid tumor formation might have a common genetic denominator. The possible role of HMG genes in growth control is further supported by results from gene targeting experiments in mice, which indicate that HMGI-C plays an important role in mammalian growth and development, as inactivation of the murine Hmgic gene resulted in the pygmy phenotype. The discovery that high mobility group protein genes might be crucial factors in the formation of uterine leiomyoma provides a new opportunity for developing alternative therapeutic strategies.

Carcinogenicity and DNA adduct formation observed in ACI rats after long-term treatment with madder root, Rubia tinctorum L.

Madder root, Rubia tinctorum L., is a traditional herbal medicine used against kidney stones. Recently we reported that lucidin, a hydroxyanthraquinone derivative present in this plant, is mutagenic in bacteria and mammalian cells. We also demonstrated the formation of DNA adducts in tissue culture and mice after treatment with this compound. To elucidate the possible carcinogenicity of madder root, three groups of male and female ACI rats received either a normal diet or a diet supplemented with 1 or 10% drug for a total period of 780 days. Weight gain and morbidity were not different among the three groups. Non-neoplastic lesions related to the treatment were evident in the liver and kidneys of both sexes. Moreover, dose-dependent increases in benign and malignant tumour formation were observed in the liver and kidneys of treated animals. 32P-post-labelling analysis showed an increase in the overall level of DNA adducts observed in the liver, kidney and colon of rats treated with 10% madder root in the diet for 2 weeks. HPLC analysis of 32P-labelled DNA adducts revealed a peak co-migrating with an adduct obtained after in vitro treatment of deoxyguanosine-3'-phosphate with lucidin. These observations suggest that the use of madder root for medicinal purposes is associated with a carcinogenic risk.

Multiple stages and genetic alterations in immortalization, malignant transformation, and tumor progression of human skin keratinocytes.

An in vitro carcinogenesis model of human skin keratinocytes has been developed based on the spontaneously immortalized keratinocyte cell line HaCaT. Immortalization, the initial stage in human carcinogenesis in vitro, was induced by ultraviolet-type mutations in the p53 gene followed by further genetic alterations leading to the loss of senescence genes, in particular on chromosome 3p. Despite multiple genetic changes, the HaCaT cell line sustained its genomic balance up to high passage levels and maintained a non-tumorigenic phenotype. Tumorigenic transformation was induced by ras oncogene transfection but also by culture stress and elevated temperature, resulting in benign and malignant tumorigenic clones. Malignant conversion was associated with the loss of a copy of chromosome 15, leading to a decrease in thrombospondin-1 (TSP-1) expression. Heat-induced malignant conversion was associated with a gain of material on chromosome 11, including the cyclin D1 gene. The microenvironment plays a major role in tumorigenic transformation and the control of malignant cells. Overexpression of platelet-derived growth factor in HaCaT cells caused mesenchyme activation and formation of benign tumors. Halting tumor angiogenesis completely prevented invasion of malignant cells and induced a benign tumor phenotype. Transfer of a normal chromosome 15 or TSP-1 transfection into a skin carcinoma line resulted in tumor suppression due to TSP-1-blocked tumor vascularization. Because of the reduced TSP-1 expression, blood vessels infiltrated the tumor, and it expanded. Progression to more aggressive tumor phenotypes required the in vivo environment and was caused by selection of a subpopulation and further genetic modifications. The improved autonomous growth of these cells was associated with new expression of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, which acted in an autocrine manner to stimulate proliferation and migration. With this in vitro skin carcinogenesis model we were able to demonstrate multiple stages in the transformation process that were associated with different genetic and phenotypic characteristics. In addition, we documented that modulation of the tumor stroma plays an important and decisive role in tumor development and progression. From this we hypothesize that the growth restraints of the microenvironment are increasingly lost with advancing stages of carcinogenesis but can be restored by modulation of the tumor stroma.

Structural analysis of Drosophila merlin reveals functional domains important for growth control and subcellular localization.

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila. As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane- associated NH2-terminal 350 amino acids of Merlin. Removal of a seven-amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene.

Less expression of cyclin E in cutaneous squamous cell carcinomas than in benign and premalignant keratinocytic lesions.

In a previous study, we showed that overexpression of cyclin D, a G1 cyclin, is frequently associated with keratinocyte carcinogenesis as an early event. Another G1 cyclin, cyclin E, was recently suggested to be a prognostic marker for breast cancer. In order to evaluate the role of cyclin E in human keratinocyte carcinogenesis, we analysed the expression of cyclin E by immunohistochemistry in normal skin, seborrheic keratosis (SK), keratoacanthoma (KA), actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Positive cells were seen rarely in normal epidermis, in 9 of 20 cases of SK, in 5 of 6 cases of KA, in 9 of 13 cases of AK and in all 27 cases of BD. Some of the cases of AK and BD had positive cells in the superficial epidermis, where atypicality is less obvious. In contrast, positive cells were seen in 4 of 25 cases of SCC and none of 15 cases of BCC. These results suggest that expression of cyclin E plays a role in the formation of benign and premalignant keratinocytic tumors, whereas down-regulation of cyclin E expression may be involved in carcinogenesis in human keratinocytes.

Mutations and polymorphisms in the tuberous sclerosis complex gene on chromosome 16.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder of benign tumor formation, hamartomata, and hamartias. TSC has been shown to be genetically heterogeneous, with one causative gene mapping to chromosome 9q (denoted TSC1) and at least one other gene on chromosome 16p (denoted TSC2). The TSC2 gene was recently cloned. We have tested 88 TSC probands with the TSC2 cDNA by Southern blotting searching for gross deletions/rearrangements/insertions. We detected two deletions and a rare intragenic polymorphic variant. This is a similar rate of mutation detection (2/88; 2.3%) to that in the original report (10/260/; 3.8%). The rare polymorphic variant was initially detected in the proband of a chromosome 9-linked multiplex TSC family. The polymorphism segregated with previously tested markers on chromosome 16 independently of the disease gene, verifying that the variation was unrelated to TSC status. We have also begun searching for subtle mutations by SSCA and direct sequencing. After screening three exons, we found two intragenic polymorphic variants. Both polymorphisms are common, making them useful for linkage studies in known affected families.

Mutations and polymorphisms in the tuberous sclerosis complex gene on chromosome 16

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder of benign tumor formation, hamartomata, and hamartias. TSC has been shown to be genetically heterogeneous, with one causative gene mapping to chromosome 9q (denoted TSC1) and at least one other gene on chromosome 16p (denoted TSC2). The TSC2 gene was recently cloned. We have tested 88 TSC probands with the TSC2 cDNA by Southern blotting searching for gross deletions/rearrangements/insertions. We detected two deletions and a rare intragenic polymorphic variant. This is a similar rate of mutation detection (2/88; 2.3%) to that in the orignial report (10/260/;3.8%). The rare polymorphic variant was initially detected in the proband of a chromosome 9-linked multiplex TSC family. The polymorphism segregated with previously tested markers on chromosome 16 independently of the disease gene, verifying that the variation was unrelated to TSC status. We have also begun searching for subtle mutations by SSCA and direct sequencing. After screening three exons, we found two intragenic polymorphic variants. Both polymorphisms are common, making them useful for linkage studies in known affected families. © 1997 Wiley-Liss, Inc.

Basic fibroblast growth factor selectively regulates ornithine decarboxylase gene expression in malignant H-ras transformed cells

Cell growth regulation by fibroblast growth factors (FGFs) is highly complex. The present study demonstrates a novel link between alterations in bFGF regulation during malignant conversion and the expression of ornithine decarboxylase, a key rate-limiting and regulatory activity in the biosynthesis of polyamines. H-ras transformed mouse 10T 1/2 cell lines exhibiting increasing malignant potential were investigated for possible bFGF-mediated changes in ornithine decarboxylase gene expression. Selective induction of ornithine decarboxylase gene expression was observed, since, in contrast to nontransformed 10T 1/2 cells and cells capable of only benign tumor formation, H-ras transformed metastatic cells exhibited marked elevations in ornithine decarboxylase message levels. Evidence for regulation of ornithine decarboxylase gene expression by bFGF at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to bFGF exposure inhibited the increase in ornithine decarboxylase message. Furthermore, striking differences in the rates of ornithine decarboxylase message decay were observed when cells treated with bFGF were compared to untreated control cells, with the half-life of ornithine decarboxylase mRNA increasing from 2.4 h in untreated cells to 12.5 h in cells exposed to bFGF. Evidence was also obtained for a cycloheximide-sensitive regulator of ornithine decarboxylase gene expression whose effect, in combination with bFGF, resulted in a further augmentation of ornithine decarboxylase gene expression. Furthermore, evidence is presented to suggest a possible role for G-protein-coupled events in the bFGF-mediated regulation of ornithine decarboxylase gene expression. The bFGF regulation of ornithine decarboxylase expression in H-ras transformed malignant cells appeared to occur independent of protein kinase C-mediated events. These results show that bFGF can modulate ornithine decarboxylase gene expression in malignant H-ras transformed cells and further suggests a mechanism of growth factor stimulation of malignant cells wherein early alterations in the regulatory control of ornithine decarboxylase gene expression are critical.

Regulation of neurofibromin expression in rat sciatic nerve and cultured Schwann cells.

Loss of function mutations at the NF1 locus may act intrinsically in Schwann cells to cause the formation of benign Schwann cell tumors (neurofibromas) in patients with type 1 neurofibromatosis. To identify contexts in Schwann cells in which such mutations may play an important role, we measured the levels of NF1 mRNA and neurofibromin in rat sciatic nerve during development, after axotomy, and in cultured rat Schwann cells. NF1 mRNA was present in developing sciatic nerve throughout the period of active Schwann cell proliferation and myelination. After nerve transection, no alteration in NF1 message level was detected, but neurofibromin levels increased, as assessed by immunohistochemistry and Western blotting, suggesting that, in vivo, neurofibromin expression in Schwann cells is post-transcriptionally induced during Wallerian degeneration. Cultured rat Schwann cells constitutively expressed NF1 mRNA and neurofibromin. Schwann cell proliferation induced by exposure to serum and forskolin was not associated with changes in NF1 mRNA or neurofibromin expression, whereas Schwann cell proliferation induced by extracts of embryonic brain membranes was associated with increased NF1 message and neurofibromin expression. Thus, Schwann cells, both in vivo and in vitro, express NF1 mRNA constitutively; the expression of NF1 mRNA and neurofibromin is modulated by only some mitogenic stimuli in Schwann cells.