The non-coding RNAs as riboregulators.

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro-behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.

Sulfhydryl group quantitation of hepatoma and liver microsomal fractions.

The relationship of sulfhydryl and disulfide groups to protein synthesis in normal and rapidly growing tissues was investigated by quantitation of sulfhydryl groups in endoplasmic reticulum and polyribosomes of normal liver and hepatomas. Stripping by ethylenediaminetetraacetate and potassium chloride of normal liver smooth and rough endoplasmic reticulum reduced by 15 percent and increased 30 percent, respectively, the sulfhydryl groups available for carboxamidemethylation by iodoacetamide. This could reflect the removal of ribosomes from rough endoplasmic reticulum with the subsequent exposure of sulfhydryl groups. Exposed sulfhydryl groups of normal mature female rat liver smooth endoplasmic reticulum were decreased to a similar degree by the stripping procedure with ethylenediaminetetra-acetate and potassium chloride when quantitated by either iodoacetamide or 4,4'-dithiodipyridine. This was not the case in young male and female rats, where the stripping procedure failed to decrease the exposed sulfhydryl groups of smooth endoplasmic reticulum. An increase in the quantity of exposed sulfhydryl groups of normal young and mature rat liver rough endoplasmic reticulum after stripping by ethylenediaminetetraacetate and potassium chloride was observed with iodoacetamide. However, when 4,4'-dithiodipyridine was used, no change could be detected. The hypothesis that smooth endoplasmic reticulum arises by degranulation of the rough endoplasmic reticulum in vivo is not supported by our sulfhydryl group quantitation of smooth endoplasmic reticulum and in vitro degranulated rough endoplasmic reticulum. A negative correlation between exposed sulfhydryl groups on the polyribosomes and the rate of growth of normal liver and of Morris hepatomas 6 and 38B suggests that the conformation of the free polyribosomal proteins could be a control factor for the rate of protein synthesis. Faster growing hepatomas also have greater quantities of sulfhydryls and disulfides.

Differentiation of choriocarcinoma cell line (JAr).

Choriocarcinoma, a highly malignant tumor arising from the trophoblast, comprises a heterogenous population of cells including cytotrophoblasts, intermediate trophoblasts, and syncytiotrophoblasts. In order to investigate trophoblast differentiation, we used centrifugal elutriation to separate cells from the JAr choriocarcinoma cell line according to their size and to further show that the resultant cell populations differ in their stage of differentiation. Two % of the cell population consists of large, multinuclear cells, which display the highest level of chorionic gonadotropin (CG) mRNAs. The increase in the CG beta mRNA with cell size is a consequence of the transcriptional mechanism, since agents which induce differentiation in JAr cells, i.e., methotrexate, increase the level of CG alpha and CG beta transcripts, cause a shift in cell size, and result in the formation of multinuclear cells. The multinuclear cells in the JAr population arise, at least partly, from kariokinesis without cytokinesis.

Inhibition of bladder carcinoma angiogenesis, stromal support, and tumor growth by halofuginone.

We have previously demonstrated that halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and cell proliferation. We investigated the effect of halofuginone on transplantable and chemically induced mouse bladder carcinoma. In both systems, oral administration of halofuginone resulted in a profound anticancerous effect, even when the treatment was initiated at advanced stages of tumor development. Although halofuginone failed to prevent proliferative preneoplastic alterations in the bladder epithelium, it inhibited further progression of the chemically induced tumor into a malignant invasive stage. Histological examination and in situ analysis of the tumor tissue revealed a marked decrease in blood vessel density and in both collagen alpha1(I) and H19 gene expression. H19 is regarded as an early marker of bladder carcinoma. The antiangiogenic effect of halofuginone was also demonstrated by inhibition of microvessel formation in vitro. We attribute the profound antitumoral effect of halofuginone to its combined inhibition of the tumor stromal support, vascularization, invasiveness, and cell proliferation.

The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene?

H19 is a paternally imprinted gene with unknown function. It is located in close proximity to the maternally imprinted IGF-2 gene on chromosome 11p15.5. In this study no consistent relationship between the expression of these two genes in clones derived from JEG-3 and JAr cell lines could be detected. Nor could a consistent relationship be detected between the expression levels of these two genes and between certain characteristic tumorigenic properties of these clones. We included in this study clones, expressing low H19 levels, which after transfection with an H19 expression construct highly expressed the H19 gene. In tumors, formed by the injection of cells of JAr or JEG-3 clones into nude mice, the H19 expression was high and irrelevant to the expression level in the cells before the injection. The same phenomenon was found for IGF-2 expression during tumorigenesis caused by cells of different JEG-3 clones and in some but not all JAr derived clones. Both H19 and IGF-2 are biallelicly expressed in all the JAr and JEG-3 clones. In summary, our observations point to the conclusion that H19 is not a tumor suppressor gene. However, its high expression in all the tumors formed after injection of cells of the JAr and JEG-3 clones, leaves its role, if any, in choriocarcinogenesis an open question.

Characterization of the imprinted IPW gene: allelic expression in normal and tumorigenic human tissues.

IPW (Imprinted gene in the Prader-Willi syndrome region) is a recently identified paternally expressed gene. Previous work has demonstrated IPW expression in the human fetus and adult, with monoallelic expression in adult lymphoblasts and fibroblasts, and in fetal tissues. To further examine the expression of IPW, a series of experiments were carried out using RT-PCR to measure IPW expression in placentae and various fetal and tumor tissues. Biallelic expression of IPW was found in testicular germ cell tumor and bladder cancer cells, suggesting loss of imprinting in the latter case. Both H19 and Insulin-like growth Factor 2 (IGF2), two additional imprinted genes, also showed biallelic expression in those same tumors that demonstrated IPW biallelic expression. Of note, the naturally occurring parthenogenetic-derived mature teratoma unexpectedly expressed large amounts of IPW. Lastly, the pluripotent embryonal cancer cell line Tera-2 expressed IPW at the same level before and after differentiation induced by retinoic acid, suggesting that this gene functions in a 'housekeeping' capacity throughout cell growth. This was in contradistinction to H19 and IGF2, both of which showed significant transcriptional upregulation after Tera-2 cell differentiation.

Unique expression patterns of H19 in human testicular cancers of different etiology.

The expression pattern of the imprinted human H19 gene was investigated in testicular cancers of different etiology, as well as in normal testicular parenchyma, parenchyma without germ cells, and adjacent to testicular germ cell tumors of adolescents and adults (TGCTs), using RNase protection analysis, mRNA in situ hybridization and reverse-transcription polymerase chain reaction. While different total expression levels were detected in spermatocytic seminomas, lymphomas, a Sertoli cell tumor and Leydig cell tumors, none showed a disturbance of monoallelic expression. Strikingly, the majority of invasive TGCTs revealed expression of both parental alleles. The total level of expression highly correlated with differentiation lineage and stage of maturation, similar to that as reported during early normal embryogenesis. Biallelic expression could also be determined specifically in testis parenchyma containing the preinvasive lesion of this cancer. We therefore conclude that within the adult testis, biallelic H19 expression is specific for TGCTs, and that the level of expression is dependent on differentiation lineage and maturation stage. This is in agreement with the proposed primordial germ cell-origin of this cancer, and might be related to retention of embryonic characteristics in TGCTs. In addition, our data argue against H19 being a tumor suppressor gene.

Isolation of ribonuclease-free polysomes from human placenta.

Human placenta is known to have a high level of polysome-bound ribonuclease which has hindered the isolation of intact polyribosomes from this tissue. We describe conditions for preparing polysomes devoid of apparent ribonuclease activity from both first trimester and term placenta. This stable preparation was achieved by utilizing buffer at low pH containing 300 mM LiCl and precipitating the polysomes chemically with 200 mM MgCl2. The yield of polysomes obtained by this procedure is 2-2.5 fold greater than that obtained by the conventional method of preparing placental polysomes. The polysomes are considered pure as judged by the ratios of absorbance 260/280 and 260/235. Moreover, the yield of polysomes obtained is greater than 95% of the tissue content and the profile of the polysomes is probably representative of the in vivo population. This is concluded from experiments in which a known amount of labelled chick polysomes was added to fresh placental tissue and the recovery of label and its distribution was analyzed.

Phosphorylation of tyrosine in cultured human placenta.

The [32P]phosphoamino acids in proteins of first-trimester and term-cultured human placentas have been separated and their relative amounts have been measured. Significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first-trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering.

A laser light source and a digital autocorrelator were employed in the study of the molecular dyanmics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg2+ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.

Possible uncoupling of the mechanochemical process in the actomyosin system by covalent crosslinking of F-actin.

It has been speculated that F-actin depolymerizes during muscle contraction. In order to test this speculation, G-actin in F-actins were immobilized by crosslinking. While this did not affect activation of myosin ATPase, superprecipitation could be abolished. Sedimentation, light scattering, electron microscope, electrophoresis and homodyne spectra measurements seem to indicate that the average size of crosslinked segments increases with time of treatment with the crosslinking agent. At the same time, the amounts of dimers and trimers decreased. It appears that crosslinking makes F-actin filaments less flexible. The possible reasons for the loss of the capability to exhibit superprecipitation are discussed.

Phosphorylation of tyrosine in cultured human placenta.

The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.

H19 expression in hepatic metastases from a range of human carcinomas.

AIMS: To investigate the expression of the imprinted oncofetal H19 gene in hepatic metastases derived from a range of human carcinomas and assess its prognostic value with the view of developing a DNA based treatment for such metastases. METHODS: Non-radioactive in situ hybridisation for H19 RNA was performed on paraffin wax embedded sections of liver biopsies or partial hepatectomy specimens, taken from 80 patients with hepatic metastases derived from carcinomas from several medical centres in Israel. The degree of expression was graded qualitatively according to the number of cells expressing H19 and the intensity of staining. The medical files were searched for demographic data and survival times before and after diagnosis of hepatic metastases. RESULTS: H19 expression was found in the hepatic metastases of 64 of 80 patients. High expression (higher staining grades) of H19 in the metastases was found in 43 of 80 patients. However, H19 expression status in the hepatic metastases did not correlate with either the length of time to development of metastasis or overall survival. CONCLUSIONS: H19 is highly expressed in more than half of hepatic metastases derived from a range of carcinomas. Thus, these metastases may be suitable candidates for H19 DNA based treatment. Further studies are needed to determine whether H19 expression has prognostic value in metastatic liver disease using larger numbers of specific subtypes of primary carcinomas.

Inhibition of matrix metalloproteinase-2 expression and bladder carcinoma metastasis by halofuginone.

Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread.

Bromo-adenosine stimulates choriogonadotropin production in JAr and cytotrophoblast cells: evidence for effects on two stages of differentiation.

8-Bromo-adenosine (8-Br-adenosine) stimulates CG production in the JAr choriocarcinoma cell line. Because production of the alpha and beta CG subunits is coupled to the differentiation state of the trophoblasts, we compared the effect of adenosine on CG synthesis in the choriocarcinoma cell line with that in cytotrophoblast cells isolated from normal human term placenta. 8-Br-adenosine stimulated CG production by 5- to 6-fold in both cell types, whereas 8-Br-guanosine had no effect, demonstrating specificity for adenine-containing derivatives. The ratio of CG alpha to CG beta production in JAr cells was 2:1 in the absence or presence of 8-Br-adenosine, whereas in cytotrophoblasts the ratio was 14:1 in controls, but decreased to 3:1 with 8-Br-adenosine. The latter is attributed to an enhanced production of the beta-sub-unit. Immunocytochemistry revealed that CG alpha and CG beta were only expressed in about 4% of JAr cells, and 8-Br-adenosine stimulated the number of positive cells 2- to 4-fold. Since 8-Br-adenosine also inhibited the division of JAr cells, the data suggest that it stimulates CG expression by promoting exit of a population of the cells from the cell cycle. In nondividing placenta-derived cytotrophoblasts, 8-Br-adenosine affects a later step in the differentiation pathway, resulting in a greater effect on the beta- than the alpha-subunit. Thus, our data further support the hypothesis that regulation of CG biosynthesis is linked to differentiation of the trophoblast.

Parallel regulation of parentally imprinted H19 and insulin-like growth factor-II genes in cultured human fetal adrenal cells.

Adjacent, parentally imprinted, insulin-like growth factor-II (IGF-II) and H19 genes are highly expressed during embryogenesis and are important for fetal growth. Human fetal adrenals express abundantly both IGF-II and H19 genes. To clarify the significance and regulation of the H19 gene, we studied its expression in fetal adrenals. In situ hybridization experiments showed H19 RNA expression throughout the fetal adrenal cortex, with slightly higher expression in the outer definitive (adult) than in the inner fetal zone. In primary cultures of fetal adrenal cells, ACTH and other activators of the protein kinase-A signal transduction pathway increased both H19 and IGF-II RNA accumulation 1.7- to 10-fold. Staurosporine, a protein kinase-C inhibitor, increased H19 and IGF-II RNA to the same extent as did ACTH. The protein kinase-C activator 12-O-tetradecanoyl phorbol-13-acetate and cytokines, tumor necrosis factor-alpha and interferon-gamma, inhibited H19 and IGF-II RNA accumulation. Transforming growth factor-beta 1 caused a decrease in levels of H19 and IGF-II RNA, whereas the IGFs caused a slight increase. Our data show parallel multifactorial regulation of H19 and IGF-II RNAs in human fetal adrenal cells. This suggests common regulatory mechanisms for these adjacent genes.

High maternal serum chorionic gonadotropin level in Downs' syndrome pregnancies is caused by elevation of both subunits messenger ribonucleic acid level in trophoblasts.

A unique product of human placenta is CG. Its concentration in maternal blood rises exponentially until 9-10 weeks' gestation, thereafter, it decreases to about 20% of the maximum, remaining constant from 16-17 until 40 weeks. High second-trimester maternal blood level indicates an increased risk for Downs' Syndrome (DS). This study's aim was to determine whether changes occur in the genetic expression of CG subunits in cultured trisomy-21 trophoblasts compared with various gestational age controls. Second-trimester trisomy-21 trophoblasts secrete 10 times more CG than gestational age-matched controls during the first day in culture: 878 (range, 235-2230) IU/g vs. 87 (range, 20-150) IU/g (P < 0.05). This high secretion closely resembles quantities secreted by first-trimester normal trophoblasts: 7500 (range, 3,850-10,000) IU/g. Both subunits' messenger RNA content are substantially increased, CG beta much more than CG alpha, although these genes are not located on chromosome 21. We conclude that at least one cause of high second-trimester maternal blood CG in DS pregnancies is a rise in alpha and beta CG messenger RNA levels in the trophoblast. We propose that at 12-14 weeks, when rapid decrease in maternal blood CG levels can be found, higher than normal values may indicate an increased risk for DS.

Parental imprinting of the human H19 gene.

It has only recently become clear that genetic imprinting plays an important role in human embryogenesis and in processes leading to the development of pediatric cancers and other human diseases. Using a unique human tissue, the androgenetic complete hydatidiform mole, we established that the maternally inherited allele of the imprinted H19 gene is expressed. Our results also show that the paternal allele of the human IGF-II gene, a gene suspected to be parentally imprinted in humans, is expressed.

The expression of the imprinted H19 and IGF-2 genes in human bladder carcinoma.

The imprinted H19 gene is highly expressed in human embryos, fetal tissues and is nearly completely shut off in adults. However, it is reexpressed in a number of tumors including bladder carcinoma, demonstrating that H19 RNA is an oncofetal RNA. Tumors induced by injection of bladder carcinoma cell lines express H19 in contrast to the cells before injection. These observations support the notion of a positive correlation between H19 expression and bladder carcinoma. Loss of imprinting of H19 and IGF-2 was observed in samples of human bladder carcinoma.