Inhibition of Ras farnesylation by lovastatin leads to downregulation of proliferation and migration in primary cultured human glioblastoma cells.

BACKGROUND: Malignant astrocytomas are the most common primary intracranial human tumors. All therapeutic approaches are limited due to their high proliferative capacity and their ability to diffusely invade the brain. Amplification of tyrosine kinase receptors and their signaling pathways have been implicated as contributing to the molecular pathogenesis of astrocytomas, providing possible new targets for therapeutic intervention. In particular, astrocytomas, although lacking oncogenic Ras mutations, have elevated levels of activated Ras. Lovastatin, an inhibitor of the beta-hydroxy-beta-methylglutary CoA reductase (HMG-CoA-reductase), is currently used to treat patients with hypercholesterolemia. In addition, it inhibits isoprenylation of several members of the Ras superfamily of proteins and therefore has multiple cellular effects including the reduction of proliferation. MATERIALS AND METHODS: In this study, we investigated the impact of lovastatin on two human glioma cell lines and on 15 primary cell cultures established from biopsies of patients with glioblastoma multiforme (GBM,) Proliferation of glioma cell lines and primary tumor cells was determined by cell counting and by using the MTT assay. The cell morphology was analyzed by staining of actin filaments with phalloidin. Apoptosis was measured using the TUNEL assay. To investigate the influence of this drug on glioma cell motility, tumor cell migration was investigated using three dimensional spheroid disintegration assays. In addition, tumor cell invasion was analyzed with a confrontational assay between tumor spheroids and rat brain aggregates. RESULTS: Inhibition of farnesyl biosynthesis using lovastatin led to a block in Ras mediated signaling, indicated by lower MAPK activity. Consequently, tumor cell proliferation was reduced up to 80%. Lovastatin appeared to decrease glioma viability by inducing apoptosis, as indicated by morphological changes and increase of TUNEL positive cells. Lovastatin acts through isoprenoid depletion, because supplementation of the media with 50-100 microM mevalonate restored all tau eta epsilon effects. Invasion of tumor cells into brain tissue was not effected while migration was reduced beta upsilon about 30-40% in cells treated with high concentrations (> or = 100 microM) of lovastatin. This was surprising because drug treatment at lower concentrations led to a disruption of the actin cytoskeleton, as indicated by Phalloidin staining. CONCLUSION: Our data strongly suggest that inhibition of elevated Ras activity by lovastatin effectively targets the MAPK and probably other signaling pathways thus offering a pharmacological based approach for a potential treatment of human astrocytomas.

Retinoids inhibit human glioma cell proliferation and migration in primary cell cultures but not in established cell lines.

OBJECTIVE: Retinoids are known to exhibit a broad spectrum of biological activities, and they participate in the onset of differentiation and the inhibition of growth in a wide variety of cancer cells. Some of these vitamin A derivatives are already in clinical use. However, data on retinoid actions in glial tumors are rather sparse. Therefore, we studied the effects of the natural retinoic acid (RA) forms all-trans-RA, 9-cis-RA, and 13-cis-RA on glioma cell lines and primary cultures from patients with glioblastomas multiforme. METHODS: Six human glioma cell lines, one rat glioma cell line, and 20 primary cultures established from biopsies from patients with glioblastomas multiforme were investigated. Tumor cell proliferation was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cell-counting assays. Random migration out of tumor spheroids was quantified using a video-morphometry system. Invasion was investigated using a confrontational coculture test system. Retinoid receptor (RA receptor [RAR]alpha, -beta, and -gamma and retinoid X receptor [RXR]alpha, -beta, and -gamma) expression status was determined using reverse transcription-polymerase chain reaction studies. RESULTS: Treatment of five human glioma cell lines with the different retinoids at concentrations up to 10(-5) mol/L produced no reduction of proliferation, using various incubation times. For one human glioma cell line (U343MG-A) and one rat glioma cell line (C6), which were previously reported to be sensitive to retinoids, we could confirm strong inhibitory effects on proliferation and clear changes in morphological features after retinoid treatment. Application of the different retinoids to low-passage primary cultures of human glioblastomas resulted in marked inhibition of proliferation (30-95%) for all tested samples. Using three-dimensional spheroid cultures, we detected retinoid-induced decreases in cell migration (24-65%). Invasion was not affected by these vitamin A derivatives. In an analysis of the expression patterns for retinoid receptors (RARs and RXRs), all primary culture samples yielded positive results for RAR gamma and RXR alpha and negative results for RAR alpha, RAR beta, and RXR gamma, whereas the results of RXR beta expression were heterogeneous among different patients. The cell lines, irrespective of their RA sensitivities, did not exhibit any major differences in receptor expression. CONCLUSION: Retinoids strongly inhibit proliferation and migration in primary cultures of human glioblastomas multiforme. Our data support a clinical trial of retinoids for the treatment of human malignant gliomas. We observed that most established cell lines were not sensitive to RA. This difference between long-term cell lines and primary cultures cannot be explained by different retinoid receptor expression patterns.

Expression of different extracellular matrix components in human brain tumor and melanoma cells in respect to variant culture conditions.

Local tumor invasion into the surrounding brain tissue is a major characteristic of malignant gliomas. These processes critically depend on the interaction of tumor cells with various extracellular matrix (ECM) components. Because only little quantitative information about expression of ECM gene products in general and expression in response to alterations of the surrounding environment is available, the present study was designed. Four human glioblastoma cell lines (U373MG, U138MG, U251MG, GaMG) as well as four human melanoma cell lines (MV3, BLM, 530, IF6) were tested with semiquantitative RT-PCR for their ability to express mRNA of different human ECM components (fibronectin, decorin, tenascin, collagen I, collagen IV, versican). In addition, two human medulloblastoma (MHH-Med 1, MHH-Med 4) and two fibrosarcoma (HT1080, U2OS) cell lines were analyzed. Cells which were grown in DMEM medium containing 10% FCS expressed most of the analyzed protein components. When the same medium, but depleted of ECM proteins by filtrating through a membrane with cut-off at > 100 kD was used, basal mRNA expression of the ECM proteins was changed in most of the examined cell lines. Using serum free conditions, most of the cell lines again showed a variation in the expression pattern of mRNA encoding for the different ECM proteins compared to the other medium conditions. Comparing different cell lines from one tumor entity or different tumor groups, ECM expression was heterogeneous with regard to the different tumor entities as well as within the entities themselves. Migration assays revealed heterogeneous responses between the different cell lines, ECM components and culture conditions, making it difficult to correlate ECM expression patterns and migratory behavior. Our results revealed that all examined cell lines are able to produce ECM proteins in vitro. This suggests that tumor cells can modulate their microenvironment in vitro which has to be taken into consideration for studies related to migration and invasion.

Dissecting glioma invasion: interrelation of adhesion, migration and intercellular contacts determine the invasive phenotype.

The invasive cellular behavior of malignant gliomas is determined by receptor mediated cell-substratum contacts and cell-cell interaction as well as cellular locomotion. This study attempts to break down the complex phenomena of the invasive process into their components of attachment to neighboring cells, aggregate formation, adhesion to matrix substratum, migration and invasion into three-dimensional cellular aggregates separately analyzed in different in vitro assay systems. Using a panel of 13 glioma cell lines, adhesion to non-specifically or merosin coated surfaces was correlated to monolayer cell migration and dissemination of tumor cells from aggregates plated on these substrates. The formation kinetics of aggregates were determined and compared to the ability of these cells to rapidly attach and form mechanically stable cell-cell contacts. The motility rates in the different assay systems as well as cell-cell attachment was correlated to invasion of re-aggregated tumor cells into fetal rat brain. A tight positive correlation was found for substrate adhesion and monolayer migration. In contrast, cell-substratum contacts had little influence on dissemination of cells out of three-dimensional aggregates and no association between monolayer migration and migration of cells out of aggregates was detected. The ability of glioma cells to rapidly form aggregates was associated with enhanced migration out of aggregates. The capacity to invade fetal rat brain aggregates was correlated with the capacity to form stable intercellular adhesion as measured in a cell-cell adhesion assay. Invasion in this system was not found to be associated with migration in monolayer or with migration out of tumor aggregates. This study highlights that current in vitro assays for invasion only represent isolated aspects of the multi-cascade process which is involved in tumor cell invasion.

Effect of synthetic matrix-metalloproteinase inhibitors on invasive capacity and proliferation of human malignant gliomas in vitro.

Glioma invasion into the surrounding brain tissue is still a major obstacle for any therapeutical approach. As in other solid tumors, matrix-metalloproteases (MMPs) have been suggested as being involved. The aim of this study was to evaluate whether the use of MMP inhibitors to target the protease-mediated invasion process could be a feasible approach. Two human cell lines (U251 and GaMG) and surgical specimens of 6 patients with malignant gliomas were grown as monolayers and spheroid cultures respectively. MMP- and u-PA-mRNA expression was investigated by semi-quantitative RT-PCR. Invasion was studied in Matrigel-coated Boyden chamber transwell assays for monolayers and in confrontation cultures of tumor spheroids with fetal rat brain aggregates in the presence of the synthetic MMP inhibitors batimastat (BB-94) and marimastat (BB-2516). Cytotoxicity/cytostatic effects of high concentrations of both compounds were assessed by growth curves, MTT assays and flow cytometry in human glioma cell lines. Batimastat and marimastat revealed a cytostatic effect at high concentrations (above 1 microM) without cytotoxicity. Both MMP inhibitors effectively reduced glioma invasion in Boyden-chamber assays at low concentrations of 0.3 microM. In confrontation cultures, concentrations of 10 microM and above were necessary to reduce invasion. This effect was observable with inter-individual heterogeneity in the patient's tumor material. MMP inhibitors effectively reduce glioma invasion, although high concentrations were required in 3-dimensional culture systems. At these concentrations, both compounds revealed a cytostatic, but no cytotoxic effect. Thus, high local concentrations of MMP inhibitors could offer a new therapeutic strategy for the treatment of gliomas.

Expression of matrix metalloproteinases in human glioma cell lines in the presence of IL-10.

Matrix metalloproteinases have been implicated to play a vital role in glioma invasion as they degrade extracellular matrix to facilitate the subsequent migration of tumor cells into the surrounding brain tissue. The cytokine Interleukin-10 (IL-10) was detected recently in glial tumors in vivo. Expression of specific IL-10 mRNA as well as blood serum levels of IL-10 in glioma patients increased with malignancy suggesting a functional role of IL-10 in glioma progression. Moreover, glioma cell migration in vitro was enhanced in the presence of IL-10. We therefore investigated the expression of the matrix metalloproteinases (MMPs) stromelysin-1 (MMP-3), 72-kDa collagenase (MMP-2), 92-kDa collagenase (MMP-9), matrilysin (MMP-7) and the human macrophage metalloelastase (MMP-12). In addition, a possible relation between exposure of glioma cells to IL-10 and invasiveness of these cells due to MMP expression was analyzed. Experiments with Matrigel coated Boyden chambers revealed a pronounced dose dependent effect of IL-10 on glioma invasiveness. The synthetic MMP-inhibitor Marimastat markedly reduced cell invasion in the Boyden chambers confirming the significance of MMPs in the process of invasion. Subsequently, the expression level of MMPs and the serine protease uPA was investigated in 7 glioma cell lines (U373, GaMG, U251, GHE, SNB19, U138 and D54) by RT-PCR. In all but one cell line no enhancement of MMP expression by IL-10 was detected. Matrilysin in U373 cells was the only protease found to be upregulated in the presence of IL-10 dependent on cell density. The present data suggest that IL-10 related effects on the invasive properties of the cell lines are not directly mediated by an upregulation of matrix metalloproteinase expression.

Effect of changes in the CD44 gene on tumour cell invasion in gliomas.

Cell adhesion is a critical factor in the multistep process of tumour invasion. CD44 is one of the cell surface adhesion molecules responsible for interaction with hyaluronic acid, a component of the CNS extracellular matrix. The aim of the present study was to demonstrate whether alterations in the CD44 gene might account for different invasive behaviour. EcoRI restriction analysis by Southern blot hybridization revealed several additional hybridization signals in tissue specimens of two out of 16 patients with glioblastoma, indicating DNA rearrangements or point mutations, respectively, within the region of the CD44 gene. Expression patterns of CD44 isoforms in these two rearranged gliomas and in 28 other patients with malignant gliomas were analysed by RT-PCR. All cases displayed only the splice variant CD44H, which acts as hyaluronic acid receptor in glioma tumour cells. Tumour cell invasion was studied with Boyden chamber assays using hyaluronic acid as ligand and functional CD44H blocking antibody. Invasion of cells derived from those gliomas carrying the rearranged CD44 gene locus was decreased by about 50% compared with gliomas without rearrangement, indicating that the altered hybridization patterns in the two glioma samples influenced CD44H mediated glioma cell invasion through hyaluronic acid in vitro. Our results on CD44 isoform expression suggest that, in contrast to other solid tumours, gliomas seem to express only the CD44 variant. Genetic alterations within the CD44 gene might alter the binding domain of the receptor and thus account for different invasive behaviour in glioblastomas.

Fast blue, a fluorescent tracer in glioma cell culture, affects cell proliferation and motility.

The azo-dye, Fast Blue (FB), initially employed for retrograde neuronal tracing is increasingly used in cell invasion and migration studies to detect living cells in monolayer and glioma tumor cell spheroid models. As yet, it is assumed that a cell stained with a tracker dye retains the characteristics of the original cell. The following experiments compared the adhesion, migration and proliferation properties of the cell lines U373 and GaMG with and without FB staining. FB staining reduced cell adhesion (P < 0.01) and proliferative activity (P < 0.01) and also had a significant inhibitory effect on cell migration (P < 0.001). From the results presented it follows that FB staining markedly influences basic cell characteristics.

Inverse correlation of cell proliferation and expression of progesterone receptors in tumor spheroids and monolayer cultures of human meningiomas.

OBJECTIVE: The progesterone receptor (PgR) can be detected in 60 to 70% of meningiomas using immunohistochemistry] in situ. Whereas in monolayer tissue cultures the PgR is only rarely expressed, we were able recently to demonstrate the preservation of the PgR in fragment spheroid cultures of meningiomas. The aim of the present study was to evaluate the stability of PgR expression in meningioma spheroids in vitro and the correlation of PgR expression and cell proliferation in spheroids and whether meningioma cells reaggregated to spheroids from monolayer cultures to reexpress the PgR again. METHODS: Tumor fragment spheroids (Weeks 1-6) and cell monolayers (Passages 1 and 3) of 15 PgR-positive meningiomas were investigated by immunohistochemistry for the expression of PgRs and their proliferative activity, as demonstrated by positivity for the proliferation-related antigen Ki-67. To study PgR reexpression in reaggregated spheroids, Northern blots were performed. In addition, a reverse transcriptase-polymerase chain reaction technique was established and evaluated in combination with immunohistochemistry. Growth of meningioma spheroids was quantified in the presence of progesterone and the specific antagonist onapristone. RESULTS: The PgR remained stable in spheroids for 6 weeks in 9 of 13 cases that were able to be evaluated. All tumor fragment spheroids exhibited a proliferation index of 5 to 40% Ki-67-positive cells. Monolayer cell cultures, on the other hand, failed to express PgRs but revealed higher proliferation indices (40-90%) to a significant extent. The detection of PgR messenger ribonucleic acid in reaggregated spheroids by means of reverse transcriptase-polymerase chain reaction correlated to the nuclear expression of PgR in immunohistochemistry. Neither progesterone nor its antagonist onapristone altered spheroid growth in vitro. CONCLUSION: The expression of the PgR in meningiomas is preserved in spheroid cultures with low proliferation indices for at least 6 weeks, whereas monolayer cell cultures with a high proliferative activity lack PgR expression. The inverse pattern of Ki-67-positive cells in the outer regions of the spheroids and PgR-expressing tumor cells in the spheroid centers leads us to the conclusion that proliferating meningioma tumor cells do not express PgRs. This might also explain why tumor cell growth in vitro was neither affected by progesterone nor by onapristone. Monolayer cell cultures can be reaggregated to spheroids, the consequence being a reexpression of PgRs and, therefore, a down-regulation of proliferation.

Transfection and dye premarking of human and rat glioma cell lines affects adhesion, migration and proliferation.

It is assumed that a cell that is transfected for any gene addition or replacement or was premarked with a cell tracker dye retains the characteristics of the original cell. The following experiments compare the original C6 rat glioma cell line with C6 cells transfected with the retroviral plasmid LacZ, and the human glioma cell lines GaMG, U373, U251, and D54 with cells stained with tracker dyes (Dil and DiO). We tested adhesion, migration and proliferation. C6 cell transfection did not affect adhesion but decreased (p < 0.05) migration. Dil staining resulted in a significant decrease (p < 0.01) in adhesion in all cell lines but U251. After DiO staining human cell lines U373 and D54 displayed a decrease in adhesion (p < 0.01) whereas U251 and GaMG cells had enhanced adhesion (p < 0.01). Dye marking of C6, GaMG and U373 cells did not alter migratory capacity. In contrast, Dil and DiO reduced migration of U251 and D54 cells (p < 0.05). There was a decrease (p < 0.01) in proliferation of the human cell lines after Dil staining. Transfection or membrane dyes can alter basic cell characteristics. The assumption that a transfected or dye marked cell is the same as the original cell but with an additional gene or the presence of a dye in the membrane is untenable.

Potential role for retinoic acid receptor-gamma in the inhibition of breast cancer cells by selective retinoids and interferons.

Retinoids are known to inhibit the growth of a wide variety of cancer cells, including breast cancer cells. Advances made in recent years in the understanding of the molecular mechanisms of retinoid action have allowed the design of retinoids with selective activities. Such selective retinoids are of particular interest, because they may reduce the number of undesirable side effects observed with natural compounds. Here, we have compared the growth-inhibitory activities of natural retinoids with various selective retinoids, including anti-activator protein (AP)-1 selective compounds on estrogen receptor-positive and -negative breast cancer cell lines. In addition, we have investigated cooperativity between selective retinoids and IFNs and have begun to analyze the pathways that these two different growth inhibitors use for antagonizing breast cancer cell proliferation. We observe that several selective retinoids can inhibit breast cancer cells as efficiently as the natural compounds. Anti-AP-1-selective retinoids are as effective as retinoic acid receptor (RAR)-beta/gamma-selective compounds. This lets us conclude that retinoid-induced inhibition of breast cancer cell growth does not require retinoid receptor transactivation. Several synthetic retinoids including anti-AP-1-selective compounds show synergism with IFNs. However, true synergism between the two different types of growth regulators was seen only when both classes of molecules were used at low concentrations. RAR-beta/gamma and anti-AP-1-selective retinoids, but not RAR-alpha-selective compounds, induced increased RAR-gamma mRNA levels. Interestingly, IFNs at elevated concentrations (100 units/ml and higher) also induced increased RAR-gamma expression. Thus, when used at high concentrations, IFNs may activate growth-inhibitory pathways overlapping with those activated by retinoids. Because increased RAR-gamma expression is induced by the two different classes of breast cancer cell inhibitors, it is likely to have an important role in controlling the growth fo these cancer cells.

Regulation of H1(0) gene expression by nuclear receptors through an unusual response element: implications for regulation of cell proliferation.

Cloning and sequence analysis of the 5'-flanking region of the human H1(0) histone gene, a differentiation-specific member of the H1 family, has revealed several potential regulatory elements. In this study, we have characterized the interactions of nuclear receptors with an unusual response element consisting of two half-sites arranged as a direct repeat with an 8-bp spacer (DR-8). Thyroid hormone receptors (TR) bind this DR-8 as homodimers and heterodimers with RXR. Retinoic acid receptors (RARs) also bind as heterodimers with RXR to the DR-8, and this binding is enhanced in the presence of retinoic acid (RA) and/or 9-cis RA. Reporter constructs containing the DR-8 allowed a several-fold induction by T3 in the presence of TRs. RAR alpha and RAR beta allowed RA-dependent transcriptional activation whereas RAR gamma mostly increased basal activity. 9-cis RA inhibited the T3 response, indicating a hormonal cross-talk among the subfamily of nuclear receptors. Two orphan receptors, COUP-TF and v-erbA, also bind the DR-8 sequence in the human H1(0) promoter. COUP-TF, which usually represses RAREs, enhances transcriptional activation through the DR-8 whereas v-erbA completely represses TR-RXR induction of the H1(0) gene. Thus, a number of signaling pathways that play important roles during development and differentiation are able to influence the transcription rate of this special H1 subtype directly through a DR-8 response element in its promoter. Because H1(0) expression levels inversely correlate with cell proliferation, our data suggest that several nuclear receptors and the v-erbA oncogene can influence cell proliferation via the regulation of H1(0) expression.

Isolation of two murine H1 histone genes and chromosomal mapping of the H1 gene complement.

The mammalian H1 histone gene complement consists of at least seven H1 protein isoforms. These include five S-phase-dependent H1 protein subtypes and two more distantly related proteins, which are expressed upon terminal differentiation (H1o) or during the pachytene stage of spermatogenesis (H1t). In the past, three replication-dependent murine H1 genes plus the H1o and H1t genes have been isolated and characterized. In this report, we describe the sequences of two more H1 genes, and we show that all five murine replication-dependent H1 genes and the H1t gene map to the region A2-3 on Chromosome (Chr) 13. This is in agreement with our previous finding that the human H1 histone gene complement maps to 6p21.3, which corresponds to the A2-3 region on the murine Chr 13. Previous reports have shown that the replication-independent H1o genes map to syntenic regions on Chrs 22 (human H1o) and 15 (murine H1o).

Role of a distal promoter element in the S-phase control of the human H1.2 histone gene transcription.

The expression of one of the human main type H1 histone genes (termed H1.2) appears to be regulated by several trans-acting factors. Upstream of consensus regulatory regions, such as the TATA-, CCAAT- and H1-box (AAACACA) sequences, a crucial control site is located between nucleotide positions -536 and -412 (relative to the ATG initiation site). Removal of this promoter portion causes in chloramphenicol acetyl transferase reporter gene constructs a loss of the S-phase control function of the H1.2 promoter in HeLa cells. Electrophoretic mobility-shift assay and DNase I footprinting analysis suggest that the H1-box variant AAACAGA is a potential control element within the distal promoter region.

Organization and expression of H1 histone and H1 replacement histone genes.

The H1 family is the most divergent subgroup of the highly conserved class of histone proteins [Cole: Int J Pept Protein Res 30:433-449, 1987]. In several vertebrate species, the H1 complement comprises five or more subtypes, and tissue specific patterns of H1 histones have been described. The diversity of the H1 histone family raises questions about the functions of different H1 subtypes and about the differential control of expression of their genes. The expression of main type H1 genes is coordinated with DNA replication, whereas the regulation of synthesis of replacement H1 subtypes, such as H1 zero and H5, and the testis specific H1t appears to be more complex. The differential control of H1 gene expression is reflected in the chromosomal organization of the genes and in different promoter structures. This review concentrates on a comparison of the chromosomal organization of main type and replacement H1 histone genes and on the differential regulation of their expression. General structural and functional data, which apply to both H1 and core histone genes and which are covered by recent reviews, will not be discussed in detail.

Effects of insulin-like growth factor II on the generation of inositol trisphosphate, diacylglycerol and cAMP in human fibroblasts.

In human fibroblasts, exogenous insulin-like growth factor-II (IGF-II) induce a rapid redistribution of mannose 6-phosphate/IGF-II receptors. To analyze the mechanism transducing the IGF-II signal the phosphoinositide hydrolysis, 1,2-diacyglycerol and cAMP formation were studied after incubation with IGFs. While IGF-I (10 nM, 30 s) increased the inositol trisphosphate formation IGF-II (10 nM, up to 10 min) failed to affect phosphoinositide hydrolysis and had neither an effect on basal concentrations of diacylglycerol containing arachidonic acid or myristic acid nor on intracellular cAMP. On the contrary, pretreatment with IGF-II for 10 min enhanced the cAMP production stimulated by bradykinin (10 nM, 3 min) by 2.5-fold whereas no additive effects of IGF-II on the increased ligand binding to the mannose 6-phosphate/IGF-II receptor in response to bradykinin were observed. These results indicate that in fibroblasts the rapid IGF-II-induced redistribution of mannose 6-phosphate/IGF-II receptors is not mediated by inositol trisphosphate, diacylglycerol or cAMP, but that IGF-II may modulate permissively other agonist-generated signals.

Differential regulation of the human H1 zero-histone-gene transcription in human tumor-cell lines.

Cloning and sequence analysis of about 2 kb of the 5' flanking region of the human H1 zero histone gene reveals several potential regulatory elements upstream of the transcribed portion of this gene. Transfection studies using the chloramphenicol acetyl transferase (CAT) gene as a reporter gene with a series of promoter deletions revealed that the expression of the H1 zero gene may depend on a complex interplay of several transcription factors, including members of the retinoic acid and/or thyroid-hormone-receptor superfamily, at the 5' flanking region of the H1 zero gene. CAT assays demonstrate varied patterns of expression and regulation in different human tumor-cell lines. The leukemia cell line HL60 does not express H1 zero mRNA and shows no CAT activity. HeLa cells strongly express the CAT gene under the control of the H1 zero promoter. Under the same conditions, HepG2 cells also transcribe the CAT gene, although at a lower rate than HeLa cells. Using different promoter-deletion clones, the CAT activity differs in HepG2 and HeLa cells in the very distal promoter region. In both cell lines, the CAT activity decreases several fold when the region between nucleotides -450 and -600 upstream of the mRNA start site is deleted. It also decreases when just the proximal portion but not the distal promoter region is deleted. In summary, the regulatory patterns of these three cell lines differ, indicating a cell-type-specific regulation of the human H1 zero-histone-gene expression.

Increased level of histone H1(0) messenger RNA in hypoxic Ehrlich ascites tumor cells.

We have investigated the expression of the H1 histone subtype H1(0) gene in Ehrlich ascites tumor cells (EAT) under varied conditions of oxygen supply. Our results show that proliferating EAT cells express H1(0) mRNA at a basal level under normoxic conditions. Severe hypoxia leads to a cessation of cell growth and causes an accumulation of cells in G1. Here, we show that the level of H1(0) histone mRNA increases within a few hours after the onset of hypoxia.

Early increase in histone H1(0) mRNA during differentiation of F9 cells to parietal endoderm.

We have isolated and characterized cDNA clones coding for the H1 histone subtype H1(0) in mouse teratocarcinoma cells. The mRNA is 2100 nt long and contains a coding sequence which is highly related to that of the human H1(0) gene. Using this cDNA as a probe, we have shown that, in comparison to undifferentiated F9 cells, differentiated F9 teratocarcinoma cells contain large amounts of H1(0) mRNA. This increase takes place very early during differentiation and does not correlate with changes in the rate of cell division. This indicates that the accumulation of H1(0) mRNA is not the result of reduced proliferation. Most likely on the contrary, the increase in the amount of H1(0) and the resulting effects on the formation of high order chromatin structures are parts of the differentiation program induced in F9 cells.