Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 954
______
Molecular Mechanisms Involved in Glioma Cell Interactions In Vitro and Studies of PDGF B Transcript Variants
BY
SUSANNE HELLER
ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000 Dissertation for the degree of Doctor of Philosophy (Faculty of Medicine) in Pathology presented at Uppsala University in 2000
Abstract
Heller, S. 2000. Molecular Mechanisms Involved in Glioma Cell Interactions in vitro and Studies of PDGF B Transcript Variants. Acta Universitatis Upsaliensis. Comprehensive summaries of Uppsala dissertations from the Faculty of Medicine 954. 70pp. Uppsala. ISBN: 91-554-4804-6 .
Glioblastoma multiforme is a malignant brain tumor characterized byheterogeneity. Interactions between heterogeneous tumor cells aresupposed to affect the behavior of a whole tumor cell population. In thisthesis an in vitro model system of clonal glioma cell lines originating fromone glioblastoma tumor was used, and the behavior of cells in cocultureswas studied and compared the behavior of cells grown separately. The results indicate the presence of two types of interactions. In one,paracrine signals acted via extra- cellular media. This was associatedwith increased growth of the whole co-culture followed by a selectiveforce driving one clone to dominance. In the other type, the cell clonesgrew side by side without signs of paracrine signalling, in a balanceresulting in an increased terminal cell density. Further investigationsfocused on mechanisms of interactions in this combination. Two cell clones were chosen, a GFAP+ and a GFAP-, for furtherexperiments. With differential display PCR it was possible to investigatetheir specific gene expression patterns. Seventeen cDNA fragments weredifferentially expressed, among them two corresponded to knowntranscription factors, ATF3 and prox-1, one to a cytoskeletal protein,a-tropomyosin. The collection also contained eight ESTs (ExpressedSequence Tags) where the corresponding genes are unknown at present.Expression of the isolated sequences were also analyzed in a panel of 12different glioma cell lines and the results illustrate the complexity of geneexpression and of tumor heterogeneity. Genes, the expression levels of which were modulated in co-cultures and/or were cell density dependent,were also identified. PDGF B is suggested to play a role in sarcomas. The gene codes for anmRNA transcript with long UTRs, parts of which are deleted in thehomologous oncogene v-sis. The UTRs of PDGF B mRNAs in humansarcomas were investigated for deletions similar to v-sis that might resultin increased protein levels. A new transcript variant was identified,lacking a 149 base region in the 3'UTR, but its presence was notassociated with increased levels of protein. Alterations in the 5'UTR werefound more likely to be associated with increased protein levels.
Key words: Tumor heterogeneity, glioma, differential display, PDGF B.
Susanne Heller, Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, University Hospital, SE-751 85 Uppsala, Sweden
ã Susanne Heller 2000
ISSN 0282-7476 ISBN 91-554-4804-6
Printed by Eklundshofs Grafiska AB, Uppsala 2000 Potius sero quam numquam Livius This thesis is based on the following papers, which are referred to in the text by their Roman numerals:
I Heller, S., Bongcam-Rudloff, E., Kastemar, M., Enblad, P. and Nistér, M. Cell Interactions are Potent Forces in Cultures of Heterogeneous Glioma Cells. Submitted
II Heller, S. and Nistér, M. The Gene Expression Patterns in Clonally-derived Human Glioma Cells are Heterogeneous and Suggest a Relation to Neural Precursors. Submitted
III Heller, S. and Nistér M. Gene Expression Patterns Affected by Co-culturing of Clonally-derived Heterogeneous Human Cell Lines. Manuscript
IV Heller, S., Scheibenpflug, L., Westermark, B. and Nistér, M. PDGF B mRNA Variants in Human Tumors with Similarity to the v-sis Oncogene: Expression of Cellular PDGF B Protein is Associated with Exon 1 Divergence, but not with a 3´UTR Splice Variant. Int. J. Cancer: 85, 211- 222 (2000).
Reprints were made with permission from the publisher. © 2000 Whiley-Liss, Inc. TABLE OF CONTENTS
SECTION I
BACKGROUND...... 7 Astrocytic tumors 7 Glioblastoma multiforme (glioblastoma) 8 Common molecular genetic changes in glioblastoma 9 Development of astrocytes and neurons 11 Intermediate filaments in cells of the nervous system 11 Glioma cell lines 14 Tumor cell heterogeneity 16 Growth factors 18 Integrins and extra-cellular matrix 20 Direct cell-cell communication 20 Differential display 21 Tropomyosin 23
AIM ...... 25
RESULTS AND DISCUSSION...... 26 Glioma cell interactions an in vitro model system (paper I) 26 Gene expression patterns in GFAP+ and GFAP- cell lines (paper II) 32 Gene expression patterns induced by co-culturing of a GFAP+ and a GFAP- cell line (paper III). 36
CONCLUSIONS...... 39 SECTION II
BACKGROUND...... 40 Platelet-derived growth factor (PDGF) 40 Homology between PDGF B and the v-sis oncogene 41 PDGF in normal and tumor cells 43 Post-transcriptional regulation of PDGF B44 PDGF B mRNA transcript variants 44 Soft tissue tumors 45
AIM ...... 48
IDENTIFICATION OF PDGF B TRANSCRIPT VARIANTS (PAPER IV) ...... 49 Splicing mechanism of RNA 49 Methods 50 Identification of a new 3´UTR splice variant 52 Relation of normal and variant PDGF B transcripts to protein levels 52
DISCUSSION...... 52
SUMMARY...... 54
ACKNOWLEDGEMENTS...... 55
REFERENCES ...... 57 SECTION I
BACKGROUND
Tumors of the central nervous system (CNS) are difficult to treat and the survival time for patients with high grade tumors is short. Much research has been performed, in an attempt to understand the molecular mechanisms behind such tumors, but several question marks remain and it is difficult to predict a useful treatment from current knowledge. Fortunately, malignant tumors of the CNS are rare. The tumor type discussed in this thesis is glioblastoma multiforme, the most malignant form of diffuse astrocytic tumors in the brain. The cell types that constitute the normal brain are of different types and their presence serves different purposes. Two major groups of cells are present, neurons and glia cells. It was long believed that neurons were the major cell type in the brain, dividing for only a short period after birth, without the possibility of renewal, and that all other cell types were merely supporting cells. Some recent reports discuss the possibility for neurons to renew in the adult brain (Johansson et al., 1999; Reynolds and Weiss, 1992). The other group of cells, glia cells include astrocytes, oligodendrocytes, ependymal cells and microglia. Astrocytes are believed to be of the same origin as neurons, i.e. to originate from neuroectodermal stem cells (Goldman, 1996; Paterson et al., 1973). Their main purpose is to support the neurons and supply them with a suitable ionic environment. Cells in this group react when the brain is injured, for example by trauma, tumors or bleeding. About 40% of the cells in the normal adult brain are thought to be astrocytes (Rutka et al., 1997). Oligodendrocytes are myelin- producing cells which surround the neurons in CNS and Schwann cells are the peripheral nervous system (PNS) counterpart of these myelinating cells. Ependymal cells cover the ventricular lining in the brain where they form a single cell layer (Bruni, 1998; Johansson et al., 1999; Lois and Alvarez-Buylla, 1993; Lu et al., 2000). Microglia are phagocytic cells in the CNS, (Ling, 1981).
Astrocytic tumors About 5-7 new cases in a population of 100,000 (0,005-0,007%) are diagnosed as diffuse astrocytomas every year (Kleihues and Cavenee, 2000). The diffuse infiltration of adjacent tissue is one of the characteristics of a diffuse astrocytoma. The tumors can arise from
7 anywhere in the CNS. The low grade forms are defined as a well differentiated tumor with growth capacity into the surrounding brain tissue and patients with this type of tumor have a survival time of 5-10 years. However, the malignant astrocytomas rarely metastasize outside the CNS as other malignant tumors do. Astrocytic tumors are divided into subgroups according to the World Health Organization (WHO) system of tumor grading, where criteria such as pleomorphism, frequency of mitosis, regional zones of necrosis and endothelial cell proliferation are taken into consideration. WHO grade I Pilocytic astrocytoma II Astrocytoma (low-grade diffuse) III Anaplastic astrocytoma IV Glioblastoma multiforme Glioblastoma multiforme tumors fulfill all these criteria, and constitute the most malignant form of astrocytic tumors. Survival times of more than two years after diagnosis are rare.
Glioblastoma multiforme (glioblastoma) Mostly adults are affected by this disease at the age of 45-70 years, and 2-3 new cases per 100.000 are diagnosed every year (Lantos et al., 1996). According to the WHO grading system, the glioblastomas contain low-differentiated neoplastic cells. The tumors are characterized by prominent vascularization or necrosis, and are often located in one of the cerebral hemispheres. Macroscopically, the tumors are highly heterogeneous, with large areas of hemorrhage and others of necrosis. The tumors are also heterogeneous on the microscopic level, with regions of palisading cells and regions with pleomorphic cells. There can be areas with a high number of proliferative cells, and still other areas with well differentiated cells. The tumors are heterogeneous too, at the genetic level where cells with various mutations, deletions or amplifications of genes are present (Jung et al., 1999; Cheng et al., 1999 Kleihues and Cavenee, 2000; Lengauer et al., 1998). Additionally, variable glial fibrillary acidic protein (GFAP) expression is seen in the tumor-derived astrocytic cells (Watanabe et al., 1997). Tumor initiation and progression occur via multistep events, whit additive effects of alterations of genes involved in proliferation pathways, differentiation pathways and apoptosis. This leads to the transformation of normal cells, resulting in abnormal cell division and cells that are able to escape apoptosis, able to survive (Newcomb, et al., 1993; Sidransky
8 et al., 1999; Collins and James, 1993). The most common of such molecular genetic events in glioblastoma are described below.
Primary and secondary glioblastoma Two different types of glioblastoma multiforme are described in the literature; primary and secondary glioblastoma, defined by their clinical characteristics. Histologically, primary and secondary glioblastoma are similar, but there are some important differences at the genetic level. People of around 55 years are mostly affected by primary glioblastoma which also has a short clinical history. In these cases tumor with the criteria of a grade IV glioblastoma appears de novo. The genetic characteristics are epidermal growth factor receptor (EGFR) gene amplification, loss of heterozygosity (LOH) of chromosome 17p or TP53 mutations are seldom seen (Lang et al., 1994). However, some gliomas diagnosed as primary glioblastomas show TP53 mutations in the absence of EGFR amplifications (Louis, 1994). Patients with secondary glioblastoma are described as having neoplasms with TP53 mutations and LOH of chromosome 17p, but with rare (6%) EGFR amplifications (Watanabe et al., 1996). The average age for diagnosis of secondary glioblastoma is lower than for primary glioblastoma and the tumor is thought to have progressed from a low grade diffuse astrocytoma where certain genetic alterations already are present, such as TP53 mutations (65%) and overexpression of PDGF _-receptor, PDGFRA_ (60%). The low-grade tumor accumulates additional genetic alterations and develops into a high grade tumor.
Common molecular genetic changes in glioblastoma TP53 The normal function of TP53 is as a tumor suppressor gene preventing cells with DNA damage to proceed through the cell cycle i.e. enter S-phase and subsequently divide. Loss of wildtype TP53 enhances the tumor progression. Mutations or inactivation of TP53 are found in a majority of human tumors, and seem to play a key role in tumor development and progression. The chromosome location for TP53 is 17p13.1 (McBride et al., 1986) and mutations are frequently found in low-grade diffuse astrocytomas as an early genetic event as well as in secondary glioblastoma. Mutation is often followed by LOH in the 17p region (Fults et al., 1992; Sidransky et al., 1992; Van Meir et al., 1994; von Deimling et al., 1992). Another way in which the p53 pathway is inactivated is via binding to murine double-minute
9 2 (MDM2), a protein the normal function of which is to regulate TP53 activity. One consequence of MDM2 overexpression is to inactivate p53 (Picksley and Lane, 1993).
EGF and EGFR The EGFR gene is the most frequently amplified oncogene in glioblastoma tumors (Fuller- Bignar 1992, Mutant Res, Wong-Vogelstein 1987 PNAS 84). Ligands for binding and activation of EGFR are EGF and transforming growth factor alpha (TGF-_). Simultaneous expression of both ligand and receptor suggests an autocrine loop of activation in glioma (Ekstrand et al., 1991). EGFR is located on chromosome 7 but amplification is often seen in the form of double-minute chromosomes, i.e. extra chromosomal elements (Fuller and Bigner, 1992; Wong et al., 1987). About one third of all tumors that are diagnosed as glioblastoma, harbor amplification of EGFR or overexepress the gene (Ekstrand et al., 1991; Ekstrand et al., 1992; Lang et al., 1994; Venter and Thomas, 1991; Wong et al., 1987).
PDGF and PDGFR PDGF and its receptors are often overexpressed in different types of astrocytomas and overexpression of PDGF is more pronounced in the more malignant tumors (Hermanson et al., 1992; Westermark et al., 1995 and references therein). This suggests the establishment of an autocrine stimulatory pathway (Hermanson et al., 1992). PDGF receptor-_ expression was found to be associated with LOH on chromosome 17p and TP53 mutations (Hermanson et al., 1996) and it was found to be present even in low-grade tumors (Hermanson et al., 1992).
Loss of chromosome 10 Loss of one copy of chromosome 10 is a common event in both primary and secondary glioblastomas and multiple regions with putative tumor suppressor genes have been identified on chromosome 10 (Karlbom, et al., 1993; Maier, et al., 1998; Steck et al., 1999). In lower grades of astrocytic tumors, LOH of chromosome 10 is found, but more rarely (Bigner and Vogelstein, 1990; James et al., 1988). The gene, PTEN, located on chromosome 10 was isolated and described by Li et al. (Li et al., 1997) and was found to be mutated in several types of tumors, brain tumors included.
10 Development of astrocytes and neurons Multipotent stem cells in the embryonic and newborn brain are the source of mature neurons as well as of astrocytes and oligodendrocytes. Stem cells are found also in the adult brain ventricular walls and in or underneath the layer of ependymal cells (Reynolds and Weiss, 1996; Lee et al., 2000). Recent studies have shown that the adult brain does indeed harbor multipotent stem cells defined as ependymal cells with the capacity to differentiate into neurons (Johansson et al., 1999) or as sub ventricular zone (SVZ) cells (Barres, 1999; Doetsch et al., 1999). In vitro studies show that cells isolated from adult mouse brain are able to undergo differentiation both to GFAP negative neuron–like cells, and to GFAP positive astrocyte-like cells (Reynolds and Weiss, 1992). However, the neuronal and astrocytic cells of the brain develop mainly during embryogenesis and for a short period after birth, but development may continue throughout life. In the embryonic brain two major pathways have been suggested to exist for early astrocytic development. Firstly, precursor cells, initially located in the SVZ, undergo differentiation during peripheral migration to other areas of the CNS and secondly, radial glial cells are thought to undergo differentiation into astrocytes. (Goldman, 1996; Tohyama et al., 1992). How is it possible to distinguish between cells of different lineages, such as glial cells and neuronal cells? In an undifferentiated multipotent cell, the intermediate filament protein, nestin, is specifically expressed. When differentiated along the neuronal lineage, the cell starts to express neurofilament while astrocytic maturation is characterized by GFAP expression (Messam et al., 2000). Nestin expression disappears in the mature cells. What signals influence the embryonic stem cell to either differentiate into a mature neuron or into a mature astrocyte? Reynold and Weiss (Reynolds and Weiss, 1992) found that proliferation of GFAP- /neuron-specific enolase negative (NSE-) cells from adult mouse brain was possible to maintain with (EGF) treatment. However, a small proportion of these cells were able to differentiate into GFAP or NSE positive cells. This study demonstrates that cells in the adult brain have the potential to differentiate into neuronal or astrocytic cells (Johe et al., 1996).
Intermediate filaments in cells of the nervous system As described above, the different normal cell types of the CNS are characterized by the expression of different intermediate filament proteins. The exact function of the intermediate filaments is still unknown, but several possible functions have been suggested in the literature, such as connecting the nuclear membrane with the outer cytoplasmic membrane
11 and involvement in cell-cell communication. Additional important roles for the cytoskeleton are to organize the cytoplasm, to stabilize the cell shape, and to contribute to cell motility (Goldman et al., 1990). Three major classes of cytoskeletal proteins have been defined in eukaryotic cells, classified according to their diameter sizes: 7 nm actin microfilaments, 8-12 nm intermediate filaments (IFs), and 24 nm microtubules (Leung et al., 1998). The following cytoplasmic IFs are expressed in mammalian CNS with high degree of cell type specificity (Herrmann and Ueli, 1998 review). 1) Neurofilament, in neurons NF-L (low molecular weight) NF-M (middle molecular weight) NF-H (high molecular weight)
2) _-Internexin, in neurons 3) Peripherin, in diverse neuronal cells 4) Nestin, in neuroepithelial stem cells and muscle cells 5) Vimentin, predominantly in mesenchymal cells 6) Glial fibrillary acidic protein (GFAP), in glial cells
1) Neurofilaments are neuron-specific, expressed both in CNS and PNS and only expressed in mature neurons.
____-Internexin is abundant in the developing nervous system (Pachter and Liem, 1985) and was first isolated from the optic nerve. 3) Peripherin is expressed mainly in the PNS and was first isolated from neuroblastoma. 4) Nestin which has the molecular weight of 220-240 kDa (Dahlstrand et al., 1992; Tohyama et al., 1992) was first cloned from a rat cDNA library by Lendahl et al., (Lendahl et al., 1990) and found to be expressed abundantly in neuroepithelial stem cells, the source of both astrocytes, oligodendrocytes and neurons. Differentiated brain cells, mature neurons and astrocytes do not express nestin. Messam et al., (Messam et al., 2000) isolated a human nestin cDNA from fetal brain, generated a polyclonal antibody and detected nestin expression in proliferating progenitor cells, as well as co-expression with vimentin in cultured cells. Co- expression of GFAP and nestin was obtained in primary cell cultures, but at a later timepoint some of the GFAP-positive cells had lost their nestin expression. These results suggest that nestin is expressed in premature cells, that the expression continues when cells differentiate
12 along the astrocytic lineage, and that co-expression of nestin and the differentiated astrocytic cell marker GFAP occurs during progression towards a fully differentiated cell. Nestin is also expressed in human brain tumor-derived cell lines as well as in human CNS tumors, mostly gliomas, and in primitive neuroectodermal tumors (PNETs), where lower levels are found (Dahlstrand et al., 1992; Tohyama et al., 1992). 5) Vimentin is an IF of molecular weight 55 kDa and the vimentin gene is located on chromosome 10p (Ferrari et al., 1986; Mathew et al., 1990). Like GFAP, the vimentin protein is phosphorylated and depolymerized during mitosis (Chou et al., 1990). Vimentin knock-out mice develop normally without any major phenotypic changes (Colucci-Guyon et al., 1994). Co-expression of vimentin and GFAP is reported to occur in cultured astrocytes (Paetau and Virtanen, 1986). Cells only expressing GFAP and not vimentin also occur (Herpers et al., 1986; Eliasson et al., 1999). For reasons that are not fully understod, glioma cells in culture express vimentin, even if they lack vimentin as primary cultures. In a previous study the expression pattern of GFAP and vimentin were confirmed in human glioma cultures, primary as well as long-term. The results showed that all of the cells in all cultures were positive for vimentin staining (Westphal et al., 1990). Vimentin expression is mainly found in cells of mesenchymal origin but it is also expressed in glia cells (Westphal et al., 1990). 6) GFAP is an IF highly specific for astrocytes. The GFAP gene is located on chromosome 17q21 (Bongcam-Rudloff et al., 1991) and codes for a 50 kDa protein.
The GFAP promoter contains sites for a large set of transcription factors, e.g. NF-_B, Sp-1, NF-1, Ap-1, Ap-2, ATF3 and cAMP-responsive element binding protein (Besnard et al., 1991; Krohn et al., 1999a; Sarid, 1991) which suggests that GFAP-gene regulation can be mediated via growth factors, hormones and antigens (Laping et al., 1994). For example, in cultured astrocytes, GFAP is regulated by cell density (Goldman et al., 1990) and by hormones stimulating cAMP production (Shafit-Zagardo et al., 1988). Like other IFs, GFAP has the ability to assemble and disassemble in a dynamic fashion, and the protein is phosphorylated in the soluble form during mitosis (Almazan et al., 1993; Inagaki et al., 1990; Nakamura et al., 1992). Knock-out studies of GFAP in mice resulted in seemingly normal embryonic development (Gomi et al., 1995; Pekny et al., 1995), but abnormal myelination was seen about 6 months after birth (Liedtke et al., 1996). The role of GFAP is not known in detail, but it is suggested to be involved in reorganization of the cytoskeleton, maintenance of myelination and cell adhesion.
13 GFAP is expressed both under normal and pathological conditions, but higher expression levels of GFAP protein are found in the normal brain and in low malignant tumors (Duffy et al., 1982; Eng and Rubinstein, 1978), while in increased astrocytic malignancy, GFAP expression is decreased (Rutka et al., 1997, and references therein). Loss of GFAP expression is also seen in cultures of glioma cells, where disappearance of their expression is found after several passages in vitro (Westphal et al., 1990) and loss of GFAP production is associated with an increase of astrocytic malignancy (DeArmond et al., 1980; Duffy et al., 1982; Jacque et al., 1979).
Fig. 1
A simplified picture of neural cell development showing the expression of the IFs nestin, GFAP and NF.
Glioma cell lines When a tumor is excised and cell lines are established from a small part of the tumor tissue, a majority of cells in the petri dish will disappear within a few weeks. After several passages, selection of cells with an immortalized phenotype occurs; these cells are able to divide indefinitely in vitro. All normal cells in the original tumor tissue, with their aging clock still functioning, will die after a defined number of cell division cycles. This is one of the most important differences between a normal and a tumor cell. The surviving tumor cells then become established as tumor cell lines in vitro.
14 Pontén and Macintyre (Pontén and Macintyre, 1968) pioneered the work of culturing glial tumor-derived cells in vitro and optimized the culture conditions. The glioma tumors are highly heterogeneous (see next chapter) and a tumor consists of several different cell clones. During tumor progression, different cell clones come and go in a dynamic manner. The time point of excision is therefore probably of importance for the composition of cells in the cell lines subsequently established.
Fig. 2
Establishment of tumor cell lines in culture. Tumor progression, the development from a normal cell to a malignant tumor with several different cell clones is illustrated. Tumor progression continues in vitro.
Most of the established cell lines from gliomas are GFAP- and fibronectin (FN)+, and it was suggested that the GFAP- cells had a mesenchymal origin (Kennedy et al., 1987; McKeever et al., 1987; Paetau et al., 1980). However, a minority of established cell lines are GFAP+ (Bigner et al., 1981), and are thereby identified as cells of the astrocytic lineage. Extensive studies of cell lines originating from the U-343 glioma tumor have been done (Nistér, 1994, and references therein; Nistér et al., 1991; Nistér et al., 1988; Nistér et al., 1987). All analyzed cell clones derived from this cell line are regarded as having the same origin, as they all carry the same chromosome 1 alteration. This alteration is also found in an uncloned part of the tumor, a finding that supports the hypothesis that all involved U-343 tumor cells are of the same origin (Nistér et al., 1987), even if there are both GFAP+ and
15 GFAP- cells among the derived cultures. Other studies have shown that original glioma tumors do consist of GFAP+ and GFAP- cells (McKeever et al., 1987) with the majority of GFAP-/FN+ cells. The report also demonstrated that a small proportion of GFAP+ cells lose their GFAP expression as early as the second passage in culture. An additional report shows that SV40 large T transformed GFAP+ cells, loose their expression of GFAP as they become transformed (Geller et al., 1988). As mentioned earlier in this thesis, these findings also support the hypothesis that loss of GFAP expression is associated with increased malignancy. This might be the result of a selection mechanism favoring GFAP- cell clones. Co-expression of the stem cell-specific marker, nestin, with GFAP is also reported (Dahlstrand et al., 1992; Tohyama et al., 1992) as well as co-expression of the neuronal marker NF-L with GFAP in the glioma-derived cell lines U-251 MG and U-373 MG (Tohyama et al., 1993). Both mRNA and protein were examined in these investigations. The authors suggested that these glioma cells keep their potential as multipotent precursor cells, able to generate both GFAP and NF-L expressing cells. Other possibilities are that the coexpression of GFAP and NF-L in the same cell is a result of in vitro culturing. Several of the in vitro grown cells become both vimentin and nestin positive. However, co-expression of NF-L and GFAP have been reported in childhood glial tumors (Bodey et al., 1991; Bodey et al., 1990). This might be a result of the chaotic character of the tumor cell. Furthermore, subclones from the cell line U-251-MG were also analyzed and were shown to express both GFAP and NF-L mRNA (Tohyama et al., 1993). If the heterogeneous in vivo situation is the result of the generation of new cell clones, the question is if this possibility is maintained in the primary in vitro cultures. Bigner et al. (Bigner et al., 1987) showed that primary glioma cell lines indeed continued to accumulate new chromosomal alterations in vitro. The fact that in vitro model systems do not always reflect the true in vivo situation has to be taken into consideration and assessment of the results of all in vitro experiments have to be done carefully.
Tumor cell heterogeneity Tumor heterogeneity is at least partly a result of genetic instability (Heppner, 1998 and references therein; Nowell, 1976) and tumor cell clones may differ in growth rate, morphology, ability to metastasize and sensitivity to therapy (Hart and Fidler, 1981). This, of course, makes the treatment of such a tumor difficult, even more so since the cell composition is able to change during the life span of the tumor.
16 The theories of the mechanisms involved in tumor heterogeneity and tumor progression were formulated by Foulds in Neoplastic Development from 1969, reviewed by Heppner and Miller (Heppner and Miller, 1998) and Nowell hypothesized that tumor cell heterogeneity is a result of genetic instability. With the genetic instability, new cell clones are established, some of which will survive and give rise to new clones during tumor progression, and some of which are unable to survive the dynamic changes in the tumor. This hypothesis is based on the assumption that the whole tumor originated from one single cell. The phenomenon of genetic instability is more recently described in a review article by Lengauer et al., (Lengauer et al., 1998). The authors describe two levels of genetic instability leading to tumor heterogeneity and tumor progression. Firstly, alterations occur at the nucleotide level, with point mutations, base pair substitutions, insertions or deletions of single nucleotides or short DNA sequences. These alterations are the results of insufficient DNA- repair systems. Secondly, alterations occur at the chromosomal level, with loss or gain of whole or parts of chromosomes (aneuploidy). The genetic instability can be regarded as the facilitating force in the creation of a heterogeneous tumor with a high number of contributing sub-clones, but in the heterogeneous tumor there seems also to be another ¨force¨ acting in the opposite direction, i.e. to minimize the tumor progression and increase the stability. Poste et al., (Poste et al., 1981) described the presence of a stabilizing force when they analyzed mouse B16 melanoma cell lines, able to metastasize to the lung after subcutaneous injection. He found that a heterogeneous cell population gave rise to lung metastases at the same rate regardless of passage number. If isolated clones from the heterogeneous cell population were injected separately instead, they metastasized more efficiently and gave rise to new cell clones. The single cell populations were then regarded as more unbalanced. The authors also showed the consequence of disturbed equilibrium by drug treatment, resulting in a few surviving drug-resistant cells giving rise to new cell clones. The population thereby became heterogeneous again, now with all involved cell clones drug-resistant. Several groups have reported the phenomenon of clonal dominance being due to the immune system (Aabo et al., 1995; Miller et al., 1988; Waghorne et al., 1988), but another theory is the involvement of growth factors. Which factors act as growth stimulators and which act as growth inhibitors in such a situation? The cellular response to a certain growth factor is not always the same. TGF-_ for example, acts either as an activating or as an inhibitory factor on different glioma cell clones (Piek et al., 1999; Rich et al., 1999).
17 Another possible cellular interaction is known as the ¨community effect¨, described by Jouanneau et al., (Jouanneau et al., 1994). Here acidic fibroblast growth factor (aFGF) was tested in a mouse model system with carcinoma cells. The investigators found that aFGF producing cells were able to affect neighboring cells resulting in tumor progression. The aFGF cells were, however, not in the majority. This suggests a ¨community effect¨ where aFGF acts as a mediator of an effect that changes the whole cell population, even though the aFGF producing cells were in minority. As seen in several reports, some of which are described here, growth factors, their receptors and the signaling pathways mediated via activated receptors, are important for cell communication, tumor heterogeneity and tumor progression. However, cells are able to communicate in other ways, for example via matrix-bound factors or via gap-junctions, in which cell to cell contact is necessary. How is the cell heterogeneity distributed in the original tumor? In a study of a low grade oligoastrocytoma (Coons et al., 1995) the tumor was divided into 38 equal parts, and every one of them was analyzed with regard to DNA content and karyotype. One of these parts was suggested to be the ¨hot spot¨ of genetic instability. Histologically, the tumor was described to be relatively homogeneous. Another explanation for why tumor heterogeneity can arise is of course that the tumor has multiple origins, but according to several reports only a minor part of the glioblastoma tumors are derived from independent origins (Batzdorf and Malamud, 1963; Russel and Rubinstein, 1995; Barnard and Geddes, 1987), a matter that can only be proven by genetic studies with molecular markers (Berkman et al., 1992; Biernat et al., 1995; Nistér et al., 1987; Vogelstein et al., 1985).
Growth factors Growth factors are polypeptides and act as soluble factors, but many of them are also able to bind ECM (Raines and Ross, 1992). Their function is to activate specific receptors on the cell surface. When the factor binds to its receptor, dimerization and phosphorylation of the receptor follows, and a cascade of molecules that are involved in the signal transduction pathway are activated, leading to the activation of specific genes. Growth factor receptors are cell-type specific and their expression is strictly regulated by a number of different conditions.
18 Growth factors are able to activate receptors on the ligand-producing cell in an autocrine manner, or to activate receptors on adjacent cells (paracrine stimulation). PDGF (platelet-derived growth factor, see also section II) was first identified as a mitogen in serum, and platelets were described as the source of PDGF (Antoniades et al., 1979; Deuel et al., 1981; Heldin et al., 1979; Raines and Ross, 1982). It is a multifunctional protein and one of its normal functions is to stimulate cell division during wound healing. Elevated mRNA and protein levels are described in several tumors (Eva et al., 1982; Smits et al., 1992; Wang et al., 1994). Gliomas express high levels of both PDGF A and B chain and their receptors, suggesting the presence of autocrine stimulation (Hermanson et al., 1992; Nistér et al., 1991). EGF binds to EGFR, which is abundantly expressed in most mammalian cell types, especially present on epithelial cells. In non-tumor cells, EGF is expressed for example, during wound healing and in tumor cells, like glioma cells EGFR is often amplified (Kleihues and Cavenee, 2000).
TGF-_ is a growth factor that belongs to the EGF family. In its soluble form the molecular weight is about 6 kDa and this mature form is generated by proteolytic cleavage of a premature form attached to the cell membrane (Pandiella and Massague, 1991). TGF-_ is normally expressed during embryonic development, and normal adult tissues express lower levels than tumor tissues (Derynck et al., 1987). TGF-_ competes with EGF for binding to
EGFR (Massague, 1983; Ullrich and Schlessinger, 1990). In tumors, TGF-_ is involved in cell motility (El-Obeid et al., 1997) and also in control of differentiation (Luettke et al., 1993)._
TGF-_ (transforming growth factor beta) is expressed not only in transformed cells, but is also secreted by platelets. The best known function of TGF-_ is to inhibit proliferation of several cell types. In gliomas, cells can be found that are either growth stimulated or inhibited as a result of treatment with TGF-_ (Rich et al., 1999). FGF is a family of growth factors with the capacity to stimulate proliferation of cells involved in forming capillaries, like fibroblasts, endothelial cells and other cells of mesenchymal origin. Several isoforms are known; two of the most important are acidic and basic FGF, also known as FGF-1 and FGF-2, where the latter has higher capacity to activate cell proliferation. IGF-1 (insulin-like growth factor 1, also called somatomedin C) binds to its receptor, IGF- IR. Both receptor and ligands are abundantly expressed under normal conditions in both fetal
19 and adult brain. High concentrations of IGF-1 are found in serum (Svoboda et al., 1980), in glioma cell lines and tissues. Further, an autocrine pathway of cell growth stimulation is suggested (Ambrose et al., 1994). IGF-1 is also suggested to inhibit apoptosis (McCubrey et al., 1991) an effect more pronounced in anchorage-independent tumor cells than in cells in contact to the ECM or in monolayer cultures (Valentinis et al., 1999).
Integrins and extra-cellular matrix Integrins are a group of receptor molecules interacting with the ECM and with other cells in direct cell-cell contact. An integrin molecule consists of two subunits, _ and _. The contact between the integrins and the intracellullar cytoskeletal filaments constitutes a way of communication from the ECM to the cytoplasm (Friedlander et al., 1996; Giese et al., 1995). The extracellular part of the receptor binds to the ECM proteins, for example fibronectin, collagen, laminin and tenascin (Uhm et al., 1999). The interactions between integrins and ECM lead to modulation of intracellular signaling pathways affecting cell proliferaton, migration and survival (Varner and Cheresh, 1996). In neoplastic astrocytes several integrins are overexpressed (Paulus et al., 1994) and invasion of tumor cells in vivo is mediated by integrins (Paulus et al., 1996; Paulus and Tonn, 1994).
Direct cell-cell communication Gap junctions form channels between cells and make it possible to connect cytoplasms for direct cell-cell communication. Gap junctions are important for normal cell proliferation and development (MacDonald, 1985) and are composed of several connexin (Cx) proteins. In astrocytes 43 connexin (Cx 43) is the most common variant (Shinoura et al., 1996) and is highly conserved during evolution (el Aoumari et al., 1990; Pitts et al., 1988). Cx 43 is expressed in almost all cell types together with other connexin proteins, but in astrocytes only Cx 43 is found. Normal astrocytes are coupled by a huge number of gap junctions to neighboring cells, while neurons and oligodendrocytes rarely use this system (Nedergaard, 1994). A first report that loss of communication via gap junctions resulted in increased cellular growth and capacity to metastasize was presented in 1966 (Loewenstein and Kanno, 1966). More recent reports describe the correlation between loss of functional gap junctions and increased metastatic potential and invasiveness (Nicolson et al., 1988), and the importance of
20 gap junctions for tumorigenesis (Yotti et al., 1979). An interesting finding was made by Zhang et al (Zhang et al., 1999). They showed that upon co-culturing, a connexin-expressing glioma cell line influenced normal astrocytes, decreasing their GFAP expression and changing their morphology. The EGF signaling pathway is suggested to regulate Cx43 and the function of gap junctions. A recent study by Warn-Cramer et al (Warn-Cramer et al., 1998) showed that EGF- activated cells have phosphorylated Cx43 and thereby inactivated communication via gap junctions. This is interesting, because many glioma cells are known to overexpress the EGF receptor. Cx43 is also suggested to act as a tumor suppressor gene. Cx43-transfected glioma cell lines show reduced cell proliferation both in vitro and in vivo (Huang et al., 1998). The knowledge of gap junctions and their involvement in increased cell growth and metastasis is of considerable interest, especially in the context of tumor heterogeneity.
Differential display The human genome codes for approximately 80,000 individual genes and the average cell expresses about 10,000 genes. The specific gene expression of a cell is the major determining factor for the properties of the cell. Tumor cells differ in one way or another from their normal counterparts in their gene expression patterns and in a heterogeneous tumor, the cells are also mutually different. How is it possible to characterize these differences in gene expression and to characterize the differences between cells? There are several established methods for identifying expressed genes, and in papers II and III in this thesis, the differential display method was used. This method was first described by Liang and Pardee (Liang and Pardee, 1992) and is a PCR-based method (DD-PCR). The principle of the method is to isolate RNA from the cells to be compared. With these RNAs in hand, cDNA is then synthesized, with primers located in the polyA tail of the mRNA. The cDNA is then used as a template for PCR, where primer pairs are designed to align with low specificity. In the commercially available kit used in this work, the 5´primers are 13 nucleotides long and are combined with three different 3´primers, which are the same as those used in the cDNA synthesis and which are 16 nucleotides long and start with G, C or A. Several 5´-primers are then combined in pairs with the different 3´-primers. This kind of PCR reaction produces hundreds of PCR products in every reaction, which are subsequently separated on a
21 denaturing polyacrylamide gel. Radioactive labeling of one of the nucleotides (A) in the PCR reactions makes it possible to visualize the PCR products. After exposure to a photographic film, bands of interest show a differential distribution in the pools of cDNAs that were to be compared, are cut out. The sizes of the generated PCR bands are between 100 and 600 base pairs and they are hopefully coding for a differentially expressed mRNA. To confirm the differential gene expression pattern the PCR products of interest are reamplified and cloned into a plasmid vector. The cDNA fragments are then used as a probes in Northern blots where RNA from the original starting material of cells to be compared, are loaded. The short DD-PCR cDNA products then need to be sequenced and aligned to the DNA public databases available, to find out which genes they correspond to.
Fig. 3a
A schematic drawing to show the procedure of the DD method. The figure illustrates one example of a primer pair generating differentially sized PCR products.
22 Fig. 3b
The DD-PCR method, from cells to sequence analysis and Northern blot confirmation.
Tropomyosin Tropomyosin is a highly conserved actin-binding protein (Lees-Miller et al., 1990). Pre- tropomyosin mRNA is spliced into different variants, generating several isoforms of the protein. The _-tropomyosin protein is present in both non-muscle cells and muscle cells, in the latter together with the troponin complex, regulating Ca2+ sensitive interactions between actin and myosin (Lees-Miller and Helfman, 1991; MacLeod and Gooding, 1988). The role of _- tropomyosin in non muscle cells is less clear, but it is known that tropomyosins together with other actin-binding proteins, such as _-actinin, are down-regulated in differentiated astrocytes
(Abd-el-Basset et al., 1991). Increased levels of high molecular weight _-tropomyosin are found in more anaplastic astrocytomas than in normally differentiated astrocytes (Galloway et al., 1990).
Furthermore, Had et al., (Had et al., 1993) demonstrated several different _-tropomyosin transcripts with specific expression patterns in neurons, astrocytes and oligodendrocytes, suggesting a regulatory function of microfilament organization in astrocytes, indicating that tropomyosins are important for the differentiation as well as the motility of these cells.
Another interesting report concerning _-tropomyosin is given by Tada et al., (Tada et al.,
23 1997), where TGF-_ was found to increase the _-tropomyosins in lung carcinoma cells. In a recent report _-tropomyosin was isolated by differential dispay and found to be increased in
TGF-__-treated C6 glioma cells (Krohn, 1999b). This finding suggests that _-tropomyosin is induced by TGF-__signalling.
24 AIM
The aim of the present study was to investigate mechanisms of tumor cell heterogeneity, how tumor cells differ, how the tumor cells interact with each other, and the signals that are involved in these interactions. Specifically, the goals were;
1. To investigate tumor cell interactions in an in vitro system of glioma cells, with respect to the nature of the signals, that were involved, such as via extra-cellular media, via extra- cellular matrix, and via direct cell-cell contact.
2. To map the glioma cell heterogeneity with respect to mRNA expression levels using differential display.
3. To identify genes, the expression levels of which were affected by interactions between tumor cells growing in co-cultures.
25 Results and Discussion
Glioma cell interactions an in vitro model system (paper I) It was our hypothesis that tumor cell interactions occur between cells in a heterogeneous tumor. Our aim was to investigate these interactions in an in vitro model system. We used clonal glioma cell lines previously described to be of the same origin, as they all carried the same chromosome 1 alteration (Nistér et al., 1987). The original tumor, U-343, a highly malignant glioblastoma multiforme was excised and divided into two parts called U-343 MGa and U-343 MG (Westermark et al., 1973). The U-343 MGa line was later subcloned and gave rise to a huge number of cell lines (Nistér et al., 1986). Four of these cloned cell lines were used in this investigation together with U-343 MGa and U-343 MG. A common characteristic of the U-343 MGa part of the tumor was that all generated cell clones were GFAP+ and FN-, just like the parental heterogeneous cell line (U-343 MGa), while the cell line from the other part of the tumor, U-343 MG, was GFAP- and FN+. The cell lines used here were U-343 MGa Cl 2:6 (Cl 2:6), U-343 MGa Cl 2:3 (Cl 2:3), U-343 MGa 31L (31L), and U-343 MGa 5L (5L), where L stands for low passage number and Cl 2 (clone 2) for a recloned cell line derived from a high passage sample of U-343 MGa. The individual cell lines differed in many ways, but the most obvious variation was in morphology and in growth rates (Nistér et al., 1986). If we combine them in co-cultures, do the cells then communicate? What types of mechanisms are involved in the communications and what kind of forces are generated from the communication between cells? These are the questions we tried to answer. Possible interactions between phenotypically different cells were investigated concerning signals mediated via extra-cellular media (such as growth factors) and via extra-cellular matrix (such as integrins and matrix-bound growth factors) in vitro. The six different cell lines were grown separately and in two and two combinations, to mimic the in vivo situation in a simplified manner, in order to be able to interpret the possible interactions that occurred as a result of co-culturing the different cell types. The primary hypothesis was that a combination of different cell types should be favorable for the cell population as a whole, i.e. the total cell number was expected to increase in co-cultures, compared to separately grown cultures, if favorable cell interactions were present. Two of the cell lines (Cl 2:6 and 31L) were transfected with the bacterial LacZ gene, which made it possible to stain co-cultures for _-galactosidase activity with a histochemical staining
26 method, and count the stained cells, i.e. LacZ+ cells. Cl 2:6 and 31L were then followed in the respective co-culture experiments over 28 days. Sparse cells (2,600 cells/cm2) of each cell line, as well as co-culture combinations of them, were seeded and grown for 28 days in media supplemented with 10% serum. Media were changed twice a week. The cultures were grown without subdividing them, so that very dense cultures were obtained by day 28. These experiments were made both with and without LacZ transfected cells. Changes were calculated with respect to growth rate and terminal cell density, when the cell lines were combined two and two together. In order to test if soluble factors were mediating signals between different cells in the co- culture, cells were grown in insert dishes, where two cell types where grown in the same media without being in contact with each other (Fig 4).
Fig.4
Cell culture in an insert dish with conditioner cells in a bottom layer.
One cell (conditioner cell) was seeded to a confluent bottom layer and the other cell line was then sparsely seeded in the insert dish and subsequently counted, after 10 days. This procedure made it possible to obtain signals mediated from the bottom layer of surplus cells, and measure how the recipient cells in the insert reacted. An additional variant of this experiment was to transfer media from one cell type to another. In this way media were harvested from sparse and dense conditioner cells. Recipient cells were then grown in these media and counted. Similar experiments with the addition of antibodies against possible secreted factors were also made. Antibodies against PDGF-AA, PDGF-BB, and TGF-_ were added to conditioned media and the recipient cells were counted. Blocking antibodies against
27 EGFR were also used to test the possible role of TGF-_,_as this growth factor binds to EGFR with high affinity. Another possible way of mediating signals between cells is via extra-cellular matrix (ECM), as ECM proteins and integrins are involved in regulating cell growth and differentiation ADDIN ENRfu (Varner and Cheresh, 1996) . Such interactions were investigated as follows: Matrix from one of the cell lines in a co-culture combination was grown separately in 10% serum until confluence was reached. Then, ECM was prepared and the other cell line was seeded very sparsely (about 100 cells in a 10 cm petri dish) on top of the matrix. Cell colonies were counted after 21 days and with this procedure it was possible to investigate if extra-cellular matrix was a benefit or not to the cells. Two of the co-culture combinations showed obvious increased cell numbers after four weeks of culturing, namely combinations of Cl 2:6 and 343 MG, and of 31L and 5L. The other combinations initially showed slightly increased cell numbers, and additionally combinations where the cell number decreased after 28 days, such as 343 MG and 31L, and 343 MGa and 31L were identified. Even though there were initially equal amounts in the two cell lines, the final ratio after 28 days of culturing, was never equal. This was also to be expected since the cell lines showed different inherent growth rates and terminal cell densities when grown separately. In several of the combinations with 31L involved, this cell line was almost lost after 28 days of co-culturing, especially in combinations with Cl 2:6, 343 MG, and 343 MGa. In sparse co-cultures, a slight increase of total cell number was obtained in the combination of 31L and Cl 2:6 (day 1-7). When this co-culture became more dense, the increase was reduced, and at this time the co-culture contained almost only Cl 2:6, as a result of clonal selection. Two models of interaction between the cells were considered as possible mediators of such a result; a strong paracrine stimulatory effect mediated from 31L to Cl 2:6, and a negative signal from Cl 2:6 to 31L. The results from the insert dish experiments with Cl 2:6 as a conditioner were that 31L cells were inhibited in growth. The autocrine stimulatory effect of 31L was seen when 31L cells were both conditioner and recipient cells, and also in experiments where conditioned media was transferred to 31L in culture. 31L expresses high levels of TGF-_. The result of blocking EGFR with antibodies showed that about 44% of the 31L self-stimulating effect was inhibited by the antibodies. This partial inhibition suggested that factors other than TGF-__were also of importance for the stimulatory effect mediated by these cells. TGF-__was a strong candidate for mediating the paracrine stimulatory signal
28 between 31L and Cl 2:6, at the same time as TGF-_ was an autocrine stimulatory factor for 31L itself. EGFR antibody has not yet been tested in other combinations of cell lines. In another type of combination (Cl 2:6 and 343 MG) a different pattern was seen in the co- culture experiments. Growing together side by side for 28 days, this combination of cells generated higher terminal cell density than when the cells were grown separately and the clonal balance was kept for a long time. None of the two cell lines were allowed to dominate the co-culture. According to experiments with insert dishes, conditioned media and matrix, no strong interaction was observed between these two cell lines.
A summary of the differences obtained between the cell lines is presented in Table. I. Table I
Cell line cell growth GFAP PDGF A PDGF B TGF-_ TGF-_ morphology rate mRNA mRNA mRNA protein protein 343 MG fibroblast slow - + - - +++++ like Cl 2:6 small fast +++ +++++ +++++ + + polygonal 31L pleomorphic intermed. + + - +++++ ++++ /astrocytic 343 MGa pleomorphic fast nd + + + - /fibroblastic Cl 2:3 small intermed. +++ ++ - - + polygonal 5L pleomorphic intermed. +++++ +++ - - + /astrocytic
Since the particular cell combination of 343 MG and Cl 2:6 generates higher terminal cell density than the separately grown cells, there must be some kind of interactions between the two cell lines when they are grown together. The possibility of decreased apoptosis, resulting in higher cell density, needs to be taken into consideration. The proportion of apoptotic cells has not yet been tested and the increased cell number might be a result of decreased apoptosis together with growth-stimulating signals. Another effect of cellular interactions is the possibility for cells in a heterogeneous population to differentiate. We noticed that the Cl 2:6 cells changed their morphology when confluence was reached; the Cl 2:6 cells formed ¨islands¨ surrounded by 343 MG cells. In the borderline areas between the two cell types, cells with long processes were found, with a morphology unlike both Cl 2:6 and 343 MG. When immunofluorescence staining for GFAP was made, some of these cells were GFAP+, thus, by definition, they were Cl 2:6 cells, but with a dramatically changed morphology. On
29 the other hand, Cl 2:6 cells growing inside these ¨islands¨, without physical contact with 343 MG cells, showed regular morphology, i.e. they were small and polygonal. Is the result of culturing Cl 2:6 together with 343 MG that Cl 2:6 adopts a more differentiated phenotype, when in direct contact with 343 MG? This phenomenon was not seen when conditioned media or insert dishes with 343 MG as the conditioner cell were tested on Cl 2:6 cells. It was seen only when Cl 2:6 cells were grown in direct cell-cell contact with 343 MG, see also paper III ADDIN ENRfu (Heller and Nistér, 2000c) . In the co-culture of Cl 2:6 and 31L cells, two mechanisms may be possible: Firstly, that the stimulatory factor produced by 31L (TGF-___and others) is diluted and consumed by Cl 2:6 when the co-culture becomes more dense after about one week, resulting in the disappearance of 31L. The second possible mechanism is that Cl 2:6 produces a growth-stimulatory factor when cultures are sparse but that in the higher concentrations obtained in dense cultures, are inhibitory to 31L. These possibilities may both result in decreased 31L cell growth, and when Cl 2:6 becomes more dense, 31L disappears. The ¨community effect¨ described by Jouanneau et al, (Jouanneau et al., 1994) is also a possible mechanism that can take place in the co- cultures, especially in the co-cultures of Cl 2:6 and 31L, where the total favorable effect is still strong after 10 days, although 31L is in minority.
Fig. 5
Illustration of the interactions between three of the cell lines, 31L, Cl 2:6 and 343 MG. Positive signal and negative signal
30 Taken together, in this paper we show that tumor cells in vitro indeed interact with each other, and we believe that this happens also in the in vivo tumor, even if we used a simplified model system here. We also show that clonal selection mechanisms are operating in the cell combinations where paracrine signals are acting, leading to dominance of one of the cell clones. In one particular combination where no major paracrine interactions were detected, the involved cell clones grew in relative balance, side by side for a longer time.
31 Gene expression patterns in GFAP+ and GFAP- cell lines (paper II) The two cell lines Cl 2:6 and 343 MG were chosen for further analyses of tumor heterogeneity. These cell lines were chosen because of their differential GFAP and fibronectin expression, and their capacity to grow side by side without any clonal selection. What other differences occur between them at the gene expression level, and what are the consequences of these differences? In paper II these two cell lines were compared in a differential display assay, where differentially expressed mRNAs were analyzed in an attempt to map their respective specific gene expression patterns. The differential display assay resulted in 25 isolated unique gene fragments, and 17 of them were clearly differentially expressed in the two cell lines, as subsequently confirmed by Northern blot analysis. When the isolated cDNA fragments were sequenced and aligned to the NCBI databases, 17 of the isolated fragments corresponded to already known genes. The remaining 8 isolated cDNA fragments corresponded to expressed sequence tags (EST) in the database (Table II). Furthermore, the gene fragments isolated with the differential display method were also analyzed in a Northern blot panel consisting of 12 different glioma cell lines, with respect to their gene expression patterns. With all these Northern blot data in hand, we asked if it was possible to group the cell lines according to their different lineages of differentiation and with these data increase the knowledge about the origin of the tumor. Is the first tumor cell a transformed neuroepithelial stem cell or is the tumor originating from an already differentiated non-proliferating cell? In an attempt to illustrate this question the mRNA expression of neurofilament (NF-L), GFAP and nestin, was analyzed in the 12 cell lines.
32 Table II The gene fragments isolated by differential display
cloned fragment corresponding gene references number sequence in database 1-1 EST - 1-2 flotillin-1 (Bickel et al., 1997) 4-2 EST - 7-2 EST - 11-3 prox-1 (Zinovieva et al., 1996) 18-1 syntrophin B2 (Ahn et al., 1996) 19-2 Mit coxII 21-1 hnRNP A2/B1 (Kozu et al., 1995) 21-2 KIAA0108 (Nagase et al., 1995) 22-2 ADP/ATP translocase (Houldsworth and Attardi, 1988) 24-1 lysylhydroxylase 3 (Passoja et al., 1998; Valtavaara et al., 1998) 26-1 P311 (Taylor et al., 2000) 32-1 thioredoxin reductase (Gasdaska et al., 1995) 36-1 ATF3 (Hai et al., 1989; Liang et al., 1996) 37-3 nuclear matrix protein 55 (nmt 55) (Dong et al., 1993; Traish et al., 1997) 38-3 ribosomal protein L27a (Frigerio et al., 1995) 42-1 EST - 46-1 pregnancy associated plasma (Haaning et al., 1996; Kristensen et al., protein-A/EST 1994) 47-1 EST - 50-2 EST - 51-1 mitochondrial genome 53-2 prostaglandin F synthase (Nagase et al., 1995; Suzuki-Yamamoto et al., 1999) 55-2 EST - 56-1 _-tropomyosin (TPM1) (MacLeod and Gooding, 1988) 57-1 EST -
Isolated gene fragments known to be expressed in the nervous system during embryonic development or in adult brain, are the cell membrane protein, flotillin-1 (Bickel et al., 1997) the transcription factor, prox-1 (Zinovieva et al., 1996) and P311. Not much is known about the P311 gene but it is abundantly expressed in adult brain (Studler et al., 1993) and a more recent report suggests it is involved in the regulation of cellular growth (Taylor et al., 2000). Genes described to be involved in transformation are prox-1, P311 (Taylor et al., 2000) and thioredoxin reductase (Gasdaska et al., 1995). Other genes of interest are hnRNP A2/B1, heterogeneous nuclear ribonucleoprotein (Kozu et al., 1995), which show higher expression by immunohistochemical staining in neurons than in glia (Kamma et al., 1999). Two transcription factors were found, prox-1 and ATF3. These genes are of special interest, since they regulate the expression of other genes. There is a high mRNA level of prox-1 in Cl 2:6, and almost none in 343 MG. This gene is normally expressed in retina and
33 lens during embryonic development (Tomarev, 1997). In another study Wigle et al., (Wigle et al., 1999) showed that inactivation of prox-1 causes abnormal cell proliferation as a result of down-regulation of cell cycle inhibitors. The other transcription factor was ATF3, also with higher mRNA levels in Cl 2:6 than in 343 MG. This transcription factor has the capacity to both activate and inactivate gene expression (Chen et al., 1994) and is an early growth response gene expressed in the G1 phase of the cell cycle (Hagmeyer et al., 1996; Hsu et al., 1992). ATF3 is expressed after serum activation of cultured cells as a response to growth factors (Chen et al., 1994). The thioredoxin reductase gene product may also be of interest in this context. This protein is an enzyme involved in the reduction of oxidized thioredoxin, which is reported to be involved in the regulation of transcription factors such as NF-_B and AP-1. Overexpression of thioredoxin has been reported in several tumors (Abate et al., 1990; Gasdaska et al., 1999). Increased levels of active thioredoxin may be a result of overexpression of thioredoxin reductase. Cl 2:6 expresses higher levels of this mRNA than does 343 MG. The Northern blot analyses of GFAP, NF-L and nestin allowed us to divide the panel of 12 glioma cell lines into five groups with regard to their IF expression pattern. 1) Cell lines with no or very low IF mRNA expression (U-178 MG, U-1242 MG and U-343 MG). 2) Cell lines with only nestin expression, indicating the most immature phenotype (U-373 MG). 3) Cell lines with both GFAP and nestin expression suggesting that these cells belong to an early glial differentiation lineage (U-343 MGa 5L, U-343 MGa Cl 2:3, U-343 MGa Cl 2:6, U- 343 MGa 31L, and U-1231 MG). 4) Cell lines with both NF-L and nestin expression indicating an early neuronal differentiation (U-1240 MG and U-563 MG). 5) Cell lines with both NF-L, GFAP and nestin expression suggesting a situation beyond the differentiating phenotypes described above (U-251 MG sp). This was also reported by Tohyama (1993). The finding that the cell lines express nestin in varying amounts indicate that these cells have an immature phenotype. They may be on their way to a differentiated phenotype but have the characteristics of a tumor cell and prefer proliferation instead of differentiation. Co- expression of genes like prox-1 and flotillin-1 with GFAP support the hypothesis that the tumor cells are immature, as prox-1 is expressed in several cell lines. This indicates that these
34 cells are in an early stage of neural differentiation, in the astrocytic differentiation lineage. The GFAP- cell line 343 MG has the highest amount of the P311 mRNA. Why is this cell line GFAP- while the others from the same tumor are GFAP+? Has this cell line lost its GFAP expression, or does it have another origin than the others? Previous results show that all cell lines from the tumor have the same chromosome 1 translocation, indicating a common origin (Nistér et al., 1987). The 343 MG cell line may be on its way to differentiate into a neural lineage. It is tempting to speculate about the origin of the heterogeneous tumor. It is possible that the origin is a transformed stem cell, since a stem cell has the capacity to generate several different cell types. The expression of ¨embryonic¨ genes in the tumor may support this theory, rather than the alternative, that the heterogeneous tumor cells arise from an already differentiated cell. The results presented in this paper show that prox-1, which is normally associated exclusively with embryogenesis is also found to be expressed in the tumor and may indicate that the tumor consists of perturbed neuroepithelial precursor cells. Prox-1 may, normally, be involved in early development and be reactivated in the tumors.
35 Gene expression patterns induced by co-culturing of a GFAP+ and a GFAP- cell line (paper III). The aim of this investigation was to analyze mRNA expression levels when growing the 343 MG and Cl 2:6 cell lines together in co-cultures compared to when they were grown separately. The design of the DD-PCR assay allowed us also to compare the gene expression patterns in sparse and dense cultures. This was of interest since we found that different types of interactions might occur in sparse and dense cell populations (Heller, 2000a). Communication that requires cell-cell contact seems to be of importance in this co-culture, since Cl 2:6 changed morphology when in contact with dense 343 MG cells. The gene expression patterns in co-cultures were investigated with the same differential display method as described in paper II, where the two cell lines were grown separately and together in co-cultures, and seeded under the same conditions. Three different time points for harvesting cells and preparing RNA were used. At day 3 after seeding, the cultures were still very sparse, at day 10 the cultures had almost reached confluence and at day 17 the cultures were very dense. With this procedure it was possible to follow the gene expression patterns of the isolated gene fragments described in paper II, over time, as well as in co-cultures. Northern blots were run with total RNA from Cl 2:6, 343 MG and the co-culture of these two cell lines. All 25 isolated cDNA fragments from paper II were then hybridized to these Northern blot filters and signals were scanned and analyzed for signal intensity. The co- culture mRNA levels where compared to an estimated control of the two separately grown clones. We know from the previous results in paper I, that the co-culture at day 3 consists of 65% Cl 2:6 + 35% 343MG cells, at day 10 of 55% Cl 2:6 and 45% 343 MG cells, and at day 17 of 60% Cl 2:6 cells and 40% 343 MG cells. Based on these data, the control levels of mRNA were calculated and then compared to the levels found in the co-cultures. We isolated ten fragments that show changes in mRNA levels, either in co-cultures compared to separately grown cell lines, or as different levels of mRNA signals depending on cell density. Table III shows the results of these Northern blot hybridizations.
36 Table III Differentially expressed unique sequences
cloned corresponding cell density differences in fragment gene dependence controls vs. number co-cultures
1-2 flotillin 1
11-3 prox-1
21-1 hnRNPA2/B1
24-1 lysyl hydroxylase 3
26-1 P 311
36-1 ATF3
46-1 pregnancy associated plasma protein- A/EST 47-1 EST
53-2 prostaglandin F synthase 56-1 α-tropomyosin
increased mRNA levels in dense cultures or in co-cultures decreased mRNA levels constant mRNA levels The most prominent difference in co-culture vs. separately grown cultures was found for mRNA levels of _-tropomyosin and P311, see also paper II (Heller, 2000b). Both genes showed higher mRNA levels in the co-cultures of the two cell lines than in the control of separately grown cells. Also the cloned fragment 47-1, corresponding to an EST sequence, showed a clear difference, but in this instance a decreased mRNA level in the co-cultures. The transcription factor, ATF3, was the gene for which the cell density dependence of expression was most obvious. A low mRNA level was detected at day 3, higher levels were found at day 10 and the highest levels were found at day 17. Additional sequences with the same expression patterns were prox-1, and the pregnancy associated plasma protein-A/EST. The opposite pattern with low expression levels in dense cultures, was found for hnRNP A2/B1. P311 also showed a slight decrease in mRNA levels in dense cultures. One of the most interesting findings was the differential expression of tropomyosin in co-cultures vs.
37 separately grown cultures. Further studies of _-tropomyosin were therefore made to confirm the differential expression at the protein level, as well.
Different transcript sizes were obtained in Northern blots hybridized with the _- tropomyosin gene fragment. Tropomyosin isoforms are described by (Lees-Miller et al., 1990; MacLeod and Gooding, 1988). The cDNA fragment isolated with the DD-PCR method showed homology to exons common for all tropomyosin isoforms and this could explain the large number of different transcripts obtained in the Northern blot analyses. The following transcripts were found: 4.7 kb, 3.6 kb, 2.3 kb and 1.1 kb together with some weak bands of very large size (> 12 kb), probably belonging to preforms of mRNA. The protein levels of tropomyosin were then analyzed by Western blot and immunofluorescence staining. The antibody was a monoclonal mouse anti-chicken gizzard _-tropomyosin antibody recognizing tropomyosins with the molecular weight of 39 and 36 kDa. However, the variations in levels of mRNA and protein were not similar. In the Western blot analyses, protein of about 39 kDa was found in the co-cultures at days 7 and 14, but not at day 21. An additional band of 43 kDa was also found, with higher amounts in 343 MG cells compared to Cl 2:6, althought 343 MG expressed very low levels of tropomyosin mRNA. Proteins of higher molecular weight have previously been described by others when using the same antibody (Had et al., 1993; Tada et al., 1997) and the authors suggested unspecific binding of the antibody. The immunofluorescence staining of co- cultured cells, as well as of separately grown cells, showed clear tropomyosin filaments, similar to actin stress fibers in sparse cells, both in Cl 2:6, 343 MG and in the co-culture. Conversely, the dense cultures of all the three kinds showed a different staining pattern with a diffuse, irregular staining without any fibers. Similar immunofluorescense observations were made by Abd-El-Basset et al., (Abd-el-Basset et al., 1991). It might be an indication that tropomyosin is involved in mediating the effect of the cell-cell communication that takes place in the co-culture, leading to balanced growth and/or differentiation. To prove this, however, will require further analysis. Taken together, the results presented in this manuscript lead to further questions. We should like to test other modulated genes described here at the protein expression level, in order to understand their role in cell interactions. The functional effects of the genes of interest, e.g. _-tropomyosin and others, have to be further investigated, for example by transfection into negative glioma cells, and then investigation into their role in co-culture experiments.
38 Conclusions Tumor cell interactions do indeed occur in vitro but the functional effects of tumor cell heterogeneity is difficult to study. In this thesis, an in vitro system of cloned cells that interact in different ways was first defined. Subsequently, a differential display assay was applied to identify differences between the cells at the mRNA level, as well as to identify changes in the gene expression patterns resulting from co-culturing. As the results indicate, tumor heterogeneity is very complex, and the behavior of one cell clone depends on the surrounding types of cells. In the true in vivo situation, normal cells are also involved in cellular communication, e.g. endothelial cells and normal non-transformed glial and neuronal cells. Their role is probably also of importance in the context of heterogeneity, but has not been investigated in this thesis. Two major situations were identified when co-culturing the cell clones in vitro. Where paracrine signals like TGF-_ were operating, the recipient stimulated cell clone was able to overgrow the culture and become dominant. The donor cell clone then disappeared probably as a consequence of a negative paracrine signal, lack of space, nutrients and/or growth promoting factors. In the other situation identified, the involved cell clones grew side by side in a balance, where no strong paracrine stimulation was found. No clone became dominant, but in this situation an increased terminal cell density was observed. With the differential display assay several gene fragments were isolated, some of them corresponding to genes not yet known to be expressed in gliomas. It is likely that we identified only a partial display of all differences in gene expression present in the tumor cells. It is notable that GFAP not was found with this method, nor any of the growth factors known to constitute the differences between Cl 2:6 and 343 MG, such as PDGF A, PDGF B,
TGF-_ and TGF-_. The results presented in this thesis provide a platform for further exciting investigations into the complexity of tumor heterogeneity.
39 SECTION II
BACKGROUND The group of tumors called soft tissue tumors, originate from extraskeletal mesodermal tissues, such as muscles, fat, fibrous tissues, blood and lymphatic vessels. Several different types of tumors, both benign and malignant, are found in this group. The malignant tumors are capable of metastasizing and require radical surgery for total removal. The incidence of malignant soft tissue sarcomas is, however, low, less than 1% of the total of all cancers, but the incidence rate increases with age for most of them. In this kind of tumor and in others, genomic instability plays a major role in tumor progression. Tumor suppressor genes such as TP53 and RB may be inactivated and other genes, such as growth factors which normally are active in embryogenesis and wound healing, may be activated, leading to uncontrolled growth. Fibroblast-derived sarcomas are the most common type of soft tissue sarcoma in adults. Previous studies describe increased levels of the growth factor platelet-derived growth factor B (PDGF B) both in tumors and tumor cell lines of fibroblastic origin. PDGF B mRNA has high similarity to the oncogene v-sis, present in the retrovirus simian sarcoma virus (SSV). It is known that the gene product p28sis of v-sis is able to induce sarcomas in newborn marmosets (monkeys) and both v-sis and PDGF B have the capacity to morphologically transform, but not immortalize human fibroblasts in culture. The fact that increased mRNA levels of PDGF B are present in some sarcomas, together with its similarity to the oncogene v-sis, led us to search for abnormal regulatory mechanisms that can explain the presence of PDGF B in tumor cells.
Platelet-derived growth factor (PDGF) Platelet-derived growth factor (PDGF), acts as a mitogen for smooth muscle cells, fibroblasts and microglia. PDGF also has a role as a chemotactic signal for these cells and for monocytes, macrophages and neutrophils in wound healing (Heldin and Westermark, 1999), as well as a role in normal embryonic development (Leveen et al., 1994; Soriano, 1994). PDGF was isolated from platelets (Antoniades et al., 1979; Deuel et al., 1981; Heldin et al., 1979; Raines and Ross, 1982) after its original identification in serum (Kohler and Lipton, 1974; Ross et al., 1974; Westermark and Wasteson, 1976). The molecule consists of two polypeptide chains
40 linked together by disulfide bonds. The two chains, A and B, which have 60% homology, form all three possible dimers, AA, AB and BB. In a recent study, a third member of the PDGF family was described, PDGF-C, which binds to and induces phosphorylation of the
PDGF _-receptor (Li et al., 2000). The PDGF tyrosine kinase receptors, receptor _ and _
(PDGFR-_ and PDGFR-_) are dimerized and activated upon ligand binding (Heldin and Ostman, 1996), which leads to the activation of a cascade of molecules involved in intracellular signal transduction. The possible ligand dimers are shown in Fig. 6, with their specific abilities to bind the different combinations of receptor molecules.
Fig. 6
The different PDGF ligands and their specific binding to the PDGF receptors.
Homology between PDGF B and the v-sis oncogene Several years ago, a pet woolly monkey suffered from a tumor, which, after histological examination was diagnosed as fibrosarcoma. The animal also suffered from myelofibrosis (Theilen et al., 1971; Wolfe et al., 1971). The tumor, which mostly was composed of fibroblast-like cells, was further analyzed, and viral particles were identified in extra-cellular spaces. The virus, an acute transforming retrovirus of primate origin, simian sarcoma virus (SSV), contained specific viral genes like gag, env, and pol, together with an insertion, created by a recombination process. The viral insert sequence called v-sis, codes for the protein p28sis (Devare et al., 1983). The nucleotide sequence of v-sis was found to be highly homologous to the PDGF B, c-sis (Doolittle et al., 1983; Robbins et al., 1983; Waterfield et al., 1983). This finding was actually one of the first bioinformatic computer alignments,
41 where an amino acid sequence database was used (Doolittle, 1981). The striking finding suggested that the virus had gained the cellular sequences of PDGF B (Waterfield et al., 1983). The PDGFB gene is highly homologous between species (Bonthron et al., 1991). The PDGF-B and the v-sis gene product p28sis, are able to transform but not immortalize human fibroblasts in vitro (Clarke et al., 1984; Gazit et al., 1984) and only cells with PDGF receptors are susceptible to transformation by p28sis or PDGF B (Beckmann et al., 1988; Leal et al., 1985). In vivo studies also revealed the transforming capacity of v-sis. When SSV was injected into newborn marmosets together with its helper virus SSAV (Simian sarcoma- associated virus), the viruses generated fibrosarcoma when injected intramuscularly, and glioma when injected intracerebrally (Deinhardt, 1980). A study made in our laboratory supports the assumption that PDGF B also is able to induce tumors such as sarcomas or gliomas when injected into newborn mice (Uhrbom et al., 1998). The cDNA sequences of PDGFB and the v-sis sequence are highly homologous, but there are some important differences. Rao and collegues cloned the PDGF B cDNA (Rao et al., 1986) and described a transcript containing 7 exons distributed over 3373 nucleotides, of which, however, only 723 nucleotides are protein coding. Notable in eukaryotic cells, is the unusually long untranslated regions (UTRs), located on both sides of the protein coding sequence. The PDGF B coding sequence was found to be flanked by a 1022 bp 5’ sequence and a 1625 bp 3’ sequence. This finding raised the question if these UTRs have an important role in the regulation of the activity of the transcript. There are other reports, where long untranslated sequences are described in eucaryotic transcripts, for example in c-myc (Battey et al., 1983), suggesting a complex control of this potent oncogene. Some important differences between the PDGF B mRNA transcript and the v-sis, have been reported. Firstly, exon 1 in PDGFB is found to be deleted in v-sis. The deletion includes the signal peptid located in the 3’ end of this exon as well as a GC-rich stretch. The signal peptide is necessary for the transport of the protein to the plasma membrane where it is subsequently secreted (LaRochelle et al., 1991). Loss of this region in the v-sis transcript is compensated for by the viral env gene (King et al., 1985). The GC-rich part of exon 1 is normally able to reduce the translation rate (Rao et al., 1988; Ratner et al., 1987). Secondly, the 3’part of exon 7 is deleted in v-sis, together with an 149 base sequence in the 5´part of exon 7. The loss of the 149 base sequence leads to the relocation of the splice pointbetween exon 6 and exon 7.
42 Furthermore, when analyzing provirus in PDGF-B-induced tumors where the normal PDGF-B chain was first injected with a helper virus (Pech et al., 1989), a 149 base deletion occurred in the noncoding 3’ end of the proviral RNA, the same deletion as in v-sis.
PDGF in normal and tumor cells Is PDGF B (c-sis) the determining factor for tumor initiation and/or progression in a human situation? Eva et al., (Eva et al., 1982) found that c-sis mRNA was expressed at high levels in tumor cell lines derived from human fibrosarcomas. In their normal fibroblast counterpart mRNA was not detectable, and the authors discussed the possibility that c-sis is involved in the transformation of these cells (Heldin and Westermark, 1999 and references therein). Increased levels of PDGF B mRNA and/or protein have been reported in tumor cells and cell lines and together with the result that v-sis has the capacity to tranform normal human fibroblasts, this suggests that PDGF B may have an important role in human tumor development. In an investigation of fresh human tumor material from different sarcomas, a correlation was found between mRNA and protein of PDGF B as shown by in situ hybridization and immunohistochemical staining. In normal connective tissue PDGF B protein levels are lower than in the tumors of the same tissues, where high levels of both mRNA and protein were found (Wang et al., 1994). Malignant tumors expressed higher levels of protein and mRNA, compared to tumors of a lower grade, but the expression of PDGF B was not limited to malignant tumors. Also some of the benign tumors showed PDGF-B expresson (Wang et al., 1994). Smits et al., (Smits et al., 1992) analyzed PDGF and its receptors in fresh material from human soft tissue tumors. They found that benign and semimalignant tumors mostly expressed PDGF _-receptors while more malignant tumors expressed PDGF __receptors. Leveen et al., (Leveen et al., 1990) described varying levels of PDGF A mRNA, PDGF B mRNA, and the receptors in several types of human sarcoma cell lines. The ligand and receptor genes are independently expressed, and in normal mesenchymal cells in culture both receptors are present at different levels (Ostman et al., 1989). In the tumor cell lines analyzed by Leveen et al., (Leveen et al., 1990), some of the cell lines showed only receptor expression. In those cases an autocrine stimulation is not possible. PDGF is also expressed in several different glioma cell lines, together with its receptors (Betsholtz et al., 1986; Nistér et al., 1991; Nistér et al., 1984; Pantazis et al., 1985) as well as in vivo, in tissue from astrocytic gliomas (Maxwell et al. 1990)
43 All these findings raise several questions about PDGF B, its regulatory mechanisms and its transforming capacity. The similarities with the onc gene v-sis, suggest an ability for PDGF B to stimulate cells to enter the cell cycle and then start to divide as part of human tumor development. The differences between v-sis and normal PDGF-B mRNA raises the specific question: Does the loss of the 5’ and 3’ UTR sequences in v-sis account for its ability to transform cells? Do these UTR elements harbor sequences necessary for controlling PDGF-B mRNA levels, and can loss of these regulatory sequences subsequently lead to increased PDGF B levels and subcellular distribution and subsequent transformation of the normal cell?
Post-transcriptional regulation of PDGF B A basal promoter activity is normally responsible for PDGF B expression, resulting in the regular mRNA transcript (Khachigian et al., 1994; Pech et al., 1989; Ratner et al., 1987). The normal transcript, has an estimated half-life of about 2.6-3.4 hours in glioblastoma cells. The shortest half-life of 1.6 hours was observed in human umbilical vein endothelial cells (HUVE) (Press et al., 1988). Transcript half-life in eucaryotic cells ranges from 10 minutes to several hundred hours (Rahmsdorf et al., 1987) with the average time of more than 10 hours for mammalian mRNAs. The PDGF B transcript copy number was estimated in high expressing glioblastoma cells at 4-10 copies per cell (Press et al., 1988), but here the authors had no relevant control, since normal glial cells in culture do not express PDGF B. The presence of AU-sequences in the UTRs might be a way of increasing the mRNA degradation rate. Several eucaryotic mRNAs that code for proto-oncogenes contain AU- sequences called, adenylate/uridylate-rich elements (AREs) (Chen and Shyu, 1995). Hence, an mRNA transcript without AU sequences is expected to be stabilized (Peltz and Jacobson 1992; Kruys et al., 1990) In the PDGF B 3´UTR, AU-sequences are present within the 149 base region that is deleted in v-sis (Bonthron et al., 1991).
PDGF B mRNA transcript variants The human gene coding for PDGF B is located on chromosome 22, spanning a 22 kb region (Dalla-Favera et al., 1982) and coding for an mRNA transcript of 3.5 kb in size, which is reported to be the regular transcript (Rao et al., 1988). Only 723 bps of the transcript are protein coding. Furthermore, several transcripts with different lengths are reported in the
44 literature; (Eva et al., 1982; Fen and Daniel, 1991; Rao et al., 1988). In a more recent study, a 2.6 kb mRNA transcript was described by Dirks et al., (Dirks et al., 1995). This transcript lacked exon 1 but had an alternative initiation site in a 90 bp long exon, exon 1a, located in intron 1. This mRNA transcript was isolated from the choriocarcinoma cell line JEG-3. The short additional exon has three start codons (ATG), of which the third initiates a new start site for the PDGF B precursor protein. Sasahara et al., (Sasahara et al., 1998) found a 3.5 kb and a 2.6 kb transcript in the developing rat brain, where the 2.6 kb transcript was suggested to be the one involved in PDGF B protein expression in the CNS. Another possibility for the PDGF B gene to be expressed at a high level is described in a recent study by Simon et al., (Simon et al., 1997). A fusion between the collagen 1A gene (COL1A1) and PDGFB was found in the human tumors dermatofibrosarcoma protuberans and giant cell fibrosarcoma. The fusion between the two genes was achieved by the translocation t(17;22)(q22;q13) and a ring chromosome was seen. PDGF-B exon 1 was deleted in these rearrangements. The transforming capacity of the chimeric DNA was proven by Greco et al., (Greco et al., 1998).
Soft tissue tumors Soft tissue tumors can be divided into several subgroups, in which both benign and malignant tumors are found. Well differentiated sarcomas are often slow growing lesions, while highly malignant sarcomas are more aggressive with undifferentiated cells. Malignant tumors also have the capacity to metastasize. The histological classification of soft tissue tumors is based on the predominant cell type, i.e. which normal adult or embryonal cells they resemble. The groups are well defined, and it is very rare that a benign tumor develops into malignancy. The cell lines and tumors investigated or discussed in paper IV belong to different histological groups described in Table IV (Enzinger and Weiss, 1994).
45 Table IV Tumor Histological group Gastrointestinal epithelioid leiomyosarcoma Smooth muscle tumors III Hemangiopericytoma malignant I-II Perivascular tumors Liposarcoma II Lipomatous tumors Malignant fibrous histiocytoma III (MFH) Fibrohistiocytic tumors Sarcoma NOS Fibromatosis Fibrous tumors
Cell line Malignant fibrous histiocytoma (U-2129) Fibrohistiocytic tumors Malignant fibrous histiocytoma (U-2197) Fibrohistiocytic tumors Osteosarcoma (U-2 OS) Extraskeletal cartilaginous and osseous tumors Synovial sarcoma (U-4 SS) Synovial tumors
Fibromatoses (tumors 11 and 20). The tumors found in this group are histologically intermediate between the benign group of fibrous lesions and the malignant group of fibrosarcomas. These tumors are capable of infiltrative growth and they often recur locally, but they do not have the capacity to metastasize. The tumors classified as fibromatoses can be divided into two main subgroubs, I – Superficial (facial) fibromatoses, and II – Deep (musculoaponeurotic) fibromatoses. Dermatofibrosarcoma protuberans. Dermatofibrosarcoma protuberans belongs to the intermediate group of fibrohistiocytic tumors, inbetween the benign group and the malignant fibrous histiocytoma group. This tumor type was not included in the tumor panel in paper IV, but is referred to because of the chromosomal translocation that characterizes this tumor (Simon et al., 1997), creating a fusion mRNA between PDGFB exon 1, and COL1A1. The tumors grow in an infiltrative fashion, and are in rare cases able to metastazie, then to lung and lymph nodes. The initiation of the disease takes place in early or mid-adult life. The tumor is more common in males, and the trunk and lower extremities are often affected. Malignant fibrohistiocytic tumors (tumors 8, 12 and 14, and cell lines U-2149 and U-2197). Malignant fibrohistiocytoma is the tumor type found in most cases of soft tissue sarcomas in adults. The lower parts of the extremities are often affected in older persons, 50-70 year of age. Men suffer from this disease more than women. The cell lines U-2149 and U-2197 both originated from one patient with several tumor recurrences of this type (Genberg et al., 1989).
46 Epithelioid smooth muscle tumors (tumors 1, 4, and 15). About 5-10% of all soft tissue sarcomas are leiomyosarcomas, tumors of smooth muscles, and a part of them belongs to the malignant forms of the epithelioid tumors found in stomach and intestines, called gastrointestinal epithelioid leiomyosarcomas. Metastases are often found in the liver and lymph nodes, with cells similar to the main tumor. These tumors are found in patients in mid or late adulthood but are rare in younger patients. Lipomatous tumors (tumor 7) Liposarcoma is the second most common sarcoma in adults and the diagnoses span from well differentiated to poorly differentiated tumors. Many liposarcomas grow slowly, but can develop into very large tumors over several years. Histologically, the mass consists of a mixture of fat cells and fibrous tissue. The survival rate is higher for patients with well-differentiated tumors, and more then 70% of the patients live 5 years after surgery. For the poorly differentiated tumors, the survival rate is much lower, less than 20%. Perivascular tumors (tumor 2). Malignant hemangiopericytoma is a rather uncommon disease affecting mostly the lower extremities. Synovial sarcoma (cell line U-4 SS). Tumors belonging to this group are highly malignant and usually arise from joints or tendon sheaths. Mostly young persons are affected, the recurrence rate is high and metastases occur in over 60% of the cases. The 5 year survival is about 50%. Osteosarcoma (cell line U-2 OS). Osteosarcoma is a highly malignant bone tumor, most frequent in males aged 10-20 years, often associated with mutations in the RB gene. In older persons the disease can arise from radiation exposure. Metastases in the lung is the most common cause of death. Sarcoma NOS (not otherwise specified) (tumor 9). This tumor was a low differentiated sarcoma, and could not be further specified.
47 Aim The specific aim of section II of this thesis was to analyze different variants of PDGF B mRNA in fresh human tumor material, mainly in material from soft tissue sarcomas. In the search for different transcript variants we used the knowledge about similarities and differences between PDGF B and the v-sis oncogene. The aim was also to relate the transcript variants to the protein levels present in the same tissues.
48 Identification of PDGF B transcript variants (paper IV) In this work, we intended to study the presence of uncommon PDGF B transcripts, and to find out if there were some rare variants of this mRNA occurring in human tumor cells. The normal mRNA has long untranslated sequences in 5´ and 3´UTRs while the v-sis lacks a part of these sequences. Going out from the structure of the v-sis oncogene, the splice point downstream of the coding sequence, between exon 6 and 7, was investigated in sarcoma material, as well as regions in the 5´UTR. A modified regulatory mechanism acting on these regions of the PDGF-B transcript was suggested to generate transcripts that were more efficiently translated. The presence of alternative transcripts in low amounts, representing more efficiently translated mRNAs, may be the reason for higher levels of expressed protein in tumors. This was investigated.
Splicing mechanism of RNA Mount (Mount, 1982) described the splice junctions in a large number of genes and the most common sequences for exon-intron boundaries. Intron-exon boundaries in eucaryotic genes are described in figure 7a and a possible mechanism for the creation of a truncated exon 7, used by v-sis, is shown in figure 7b.
Fig. 7a
Intron-exon boundaries showing the specific sequences commonly found at splice-points.
49 Fig. 7b
Two possible ways to splice the PDGF B pre RNA at the exon 6/exon 7 boundary. 1) using the regular splicepoint, 2) using a cryptic splicepoint.
Methods To test the hypothesis that alternatively spliced mRNA in UTRs of PDGF B do occur, analyses were made by PCR to reveal if a deletion of the 149 bases located in the 3’UTR had occurred, lacking a part of exon 7. The PCR assay was designed with primers located on both sides of the sequence of interest (Fig 7b), and the identity of the PCR products were then confirmed by Southern blot hybridizations, where the oligonucleotide probes used for hybridization distinguished between transcripts with or without the 149 base deletion. Further analyses of the 5´UTR of the mRNA as well as of the genomic sequence in this region were also performed. A set of PCR reactions were designed to determine if the transcripts lacked the first exon, and if an alternative exon, 1a, was present. PDGF B transcripts lacking the first exon have previously been described by Fen and Daniel (Fen and Daniel, 1991) as well as transcripts with an additional exon named 1a (Dirks et al., 1995).
50 PCR reactions with a set of three different primers were used. One primer was located in exon 1 and another in exon 5. A third primer was chosen in exon 2, as this exon was supposed to be present in all transcripts. This PCR reaction should reveal the presence or otherwise of exon 1. The primer combinations used are shown in Fig. 8.
Fig. 8
PCR for detection of mRNA divergence in the 5´end of PDGF B.
The primers located in the 5´part of the mRNA transcript did not define the absolute start site of the transcripts. Hence, additional experiments were performed and 5´RACE (rapid amplification of cDNA ends) was used to identify the 5´start sites in the sarcomas and in cell lines expressing high levels of protein. Unfortunately, this method failed to give any further information, most probably due to the complex 3D structure of the transcript. Protein was detected with three different methods. Immunostaining with a monoclonal antibody against PDGF-BB was used on cryo-sectioned frozen tumor samples, as well as on frozen cell pellets from control cells and tumor cell lines. This antibody did not cross-react with PDGF-AA, as the control experiments revealed. For cells in culture two additional protein detection methods were used; a commercial radioimmunoassay (RIA) and an immunoprecipitation assay with a polyclonal PDGF-BB antibody using 35S labeled cells. The three methods together made it possible to estimate PDGF B protein levels in the cells.
51 Identification of a new 3´UTR splice variant In the Northern blot analysis, two PDGF B transcripts were discernable. The most abundant was the regular 3.5 kb transcript, visible in all samples where signals were found and in these samples an additional smaller transcript was often found. As expected, one exception was the choriocarcinoma cell line JEG-3, showing only a 2.6 kb transcript. This cell line was included as a control for this transcript variant. The PCR results indeed showed a transcript shorter by 149 bases present in most of the samples from fresh tumors as well as from the cell lines; exactly the same bases as in v-sis were missing. The transcripts were sequenced to confirm the new splice point. Such small deletions are not detectable in Northern blot analysis. The –149 base transcript was, however, never in the majority in the human cells and the regular transcript containing the 149 base fragment was dominant in all samples. In the 5´UTR the alternative exon 1a was only present in the JEG-3 cells, but exon 1 divergence occurred in several of the tumor cells and cell lines, as the 5´UTR PCR revealed.
Relation of normal and variant PDGF B transcripts to protein levels Protein levels were estimated by immunohistochemistry analysis, and were compared to the different mRNA transcript levels. No correlation between the regular transcript (+149 base) and protein levels was found, or for the alternatively spliced 3´UTR variant mRNA (-149 base) compared to expressed protein. When comparing the divergence in exon 1 and protein levels, two of the tumors (2, 20) showed high levels of protein, even higher than in the JEG-3 cells. Of note is that the alternative exon 1a was only present in the JEG-3 cell line.
Discussion The PDGFB gene coding for the PDGF B protein is under transcriptional control but the v-sis homologue has lost at least one of these control mechanisms since the 5´UTR GC rich sequence is missing. The protein product has transforming activity and it is necessary for the normal cell to strictly control expression of the protein. If the capacity to regulate gene expression is in some way altered, the cell can be transformed, as shown by in vitro studies (Clarke et al., 1984; Gazit et al., 1984), leading to uncontrolled cell proliferation. Thus, rearrangements located in the 3´ and 5´UTR regions of PDGF B, may strongly influence the translation activity and indirectly the transforming potential.
52 The results of the present investigation show that the deletion of the 149 base sequence located in the 3’UTR did not correlate to protein levels in the human tumors and cell lines investigated in this study. Samples with variations in the 5´UTR, with a small proportion of PDGF B transcripts with a putative loss of exon 1, showed higher amounts of protein. These transcripts might, as in the JEG-3 cells, have lost the function of translation-inhibitory GC sequences located in the first exon. The higher amount of protein in those samples might be explained by a small fraction of transcripts with a much more efficient translation to protein. Similar findings were reported by Sasahara et al., (Sasahara et al., 1998), where a 2.6 kb transcript lacking exon 1 was characterized in developing rat brain, generating more protein than the regular counterpart. However, a definite conclusion can not be drawn, while several tumors with high protein levels did not have the exon 1 divergence, and while we could not identify the exact 5´ sequence of these putative transcripts. A translocation of chromosomes 17 and 22, creating a fusion mRNA transcript of COL1A1 and PDGF B mRNAs lacking PDGF B exon 1, was detected in the tumors dermatofibrosarcoma protuberans and giant cell fibroblastoma (Simon et al., 1997). This may be an efficient way for the PDGF B transcript to escape the translation-inhibiting influence of the GC-rich sequence. However, no such rearrangement was found in this investigation, indicating that the COL1A-PDGFB fusion is highly specific for these fibroblast-derived tumors of intermediate malignancy. One explanation for the presence of the 149 base-deleted mRNA variant in v-sis is that when the simian sarcoma virus has the ability to chose between two transcripts coding for exactly the same protein product, it will most probably choose the shorter variant.
Future perspectives We have not identified the exact mechanism behind the higher protein levels in tumor cells, but with the results presented here there are reasons to focus on the 5´UTR in the future. For PDGF B there is no direct correlation between mRNA and protein levels. This has to be taken into consideration when analyzing tumors and cell lines, and a reliable protein detection method rather than RT-PCR or Northern blot analyses ought to be used, for example, in clinical studies.
53 Summary
A PDGF B mRNA transcript lacking a 149 base sequence normally located downstream of the protein coding sequence in the 3´UTR was identified. This variant was present as a small amount of the total PDGF B mRNA in both human tumor cell lines and fresh tumor tissues. This transcript variant was a result of using a cryptic splice site within exon 7. No correlation between the presence of transcripts lacking 149 bp in the 3’ UTR and protein levels was seen. No rearrangements were found in the PDGFB gene, but a polymorphism was found close to the exon 1 region. This polymorphism had no correlation to the sarcomas, as it was also found in normal cells from the same patients. A small proportion of transcripts with putative deletions in exon 1 was found and in some of these cases a high amount of protein was present.
54 Acknowledgements
This thesis has been carried out at the Department of Genetics and Pathology, Uppsala University. The work was supported by a grant from the Swedish Cancer Foundation. I wish to express my sincere gratitude to everyone who has supported me in my work with this thesis and especially to:
Monica Nistér, my supervisor, for her never-ending enthusiasm for my projects, for helping me gain some understanding of the world of tumor biology, and for her inexhaustible patience and also for her wild ideas.
Bengt Westermark, for inspiring seminars, great knowledge in tumor biology and for good advises.
Marianne Kastemar, Master of Cell Culturing, for skilful work in the cell hood and teaching me all about cells in culture, for her friendship and nice afternoons with the horses.
My co-authors, Lena Scheibenpflug, Erik Bongcam-Rudloff and Per Enblad, for their professional contributions.
Ulrica Westermark, for all the interesting and important discussions we have had, at the lab and at conferences, for her clarity of vision, support and last but not least, for her valuable file of methods.
Inga Hallin, for all beautiful immunostainings.
Göran Hesselager, Guo Zhongmin, Thomas Strömberg and Eva Hellmén for valuable scientific discussions and to Gijs Afink, for constructive criticism.
All members in the MN, NEH and BW groups for creating such a nice atmosphere.
55 Monica Pettersson, Jan-Ingvar Jönsson and Peter Karlberg for valuable discussions about differential display.
Gudrun Bäckström and Carl-Henrik Heldin for kindly providing antibodies and Nils-Erik Heldin for finding the plasmids I needed.
Teresita Diaz de Ståhl, Jörgen Dahlstöm and William Schannong for always giving me help with the computers when I needed it.
Frank Bittkowski for all printed microscope pictures.
Alan McWhirter, for linguistic correction.
Lene Uhrbom, Ulrica Westermark, Monica Pettersson, Kicki Andersson, Lotta Eklöf and the pre members Mozhgan Afrakthe, Christina Karlsson and Sigrídur Valgeirsdóttir, for late night poker games with dinners and pep talk.
Zhang Xiaoqun and Adila Elobeid, for always having been there and for their friendship, generosity and support!
Anna-Karin Olsson, for support and discussions at any time around the clock.
To my parents, Kerstin and Lennart, and all my Friends, without whom this work could not have been done.
Finally but most important of all, my family, Kalle, Henrik and Kristian for always believing in me and supporting me in every possible way.
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