METHOD FOR REGULATING EXPRESSION GENES
FIELD OF THE INVENTION
The present invention generally concerns methods for the regulation of gene expression, and more specifically concerns methods for the regulation of gene expression for therapeutical purposes.
BACKGROUND OF THE INVENTION
The term "angiogenesis" (also referred to at times as "neovascularization") is a general term used to denote the growth of new blood vessels both in normal and pathological conditions.
Angiogenesis is an important natural process that occurs during embryogenesis, and in the adult healthy body in the process of wound healing, and in restoration of blood flow back into injured tissues. In females, angiogenesis also occurs duiing the monthly reproductive cycle to build up the uterus hning and to support maturation of oocytes during ovulation, and in pregnancy when the placenta is established, in the process of the establishment of circulation between the mother and the fetus. The healthy body controls angiogenesis through the interactions of angiogenesis-stimulatrng growth factors, and through angiogenesis inhibitors, and the balance between the two deteπnines whether angiogenesis is turned "on" or "off1.
The following is a table that summarizes some of the known angiogenesis stimulators and inhibitors divided by their mode of action into: growth factors, proteases, trace elements, oncogenes, signal transduction enzymes, cytokines and endogenous modulators:
In the therapeutic field, there has been in recent years a growing interest in the control of angiogenesis. By one aspect, the aim was to control or diminish excessive and pathological angiogenesis that occurs in diseases such as cancer, diabetic blindness, and age related macular degeneration, rheumatoid arthritis, psoriasis, and some additional 70 conditions. In these pathological conditions the new blood vessels feed the diseased tissue, for example the tumor tissue, providing it with essential oxygen and nutrients thus enabling its pathological growth. In addition the pathological angiogenesis many times destroys the normal tissue. Furthermore, the new blood vessels, formed for example in the tumor tissue, enable the tumor cells to escape into the circulation and metastasize in other organs. Typically, excessive angiogenesis occurs when diseased cells produce abnormal amounts of angiogenetic growth factors, overwhelming the effect of the natural angiogenesis inhibitors present in the body.
Anti-angiogenetic therapies developed today, are aimed at helping new blood vessel growth through the targeting and neutralization of any of the stimulators stipulated above.
A contrasting indication of regulating angiogenesis is to encourage production of neovascularization in conditions where insufficient angiogenesis occurs. Typically, these conditions are diseases such as coronary artery diseases, stroke, and delayed wound healing (for example in ulcer lesions). In these conditions, when adequate blood vessels growth and circulation is not properly restored, there is a risk for tissue death due to insufficient blood flow. Typically, insufficient angiogenesis occurs when the tissues do not produce adequate amounts of angiogenetic growth-factors, and therapeutic angiogenesis is aimed at stimulating new blood vessels' growth by the use of growth factors or their mimics.
The main goal of the angiogenesis therapy is to produce a biobypass - i.e. to physically bypass diseased or blocked arteries, by nicking the body into building new blood vessels.
There are two main strategies for controlling angiogenesis both for anti- angiogenesis and for neovascularization purposes. The first strategy is by the
administration of the angiogenesis products (proteins) to the body, or by the administration of agents that can neutralize the angiogenesis products such as antibodies. The second approach utilizes gene transfer techniques and involves the administration of coding sequences capable of expression constructs comprising sequences coding for pro- or anti- angiogenesis products. (Refs. 4 and 5).
Clinical trials directed to gene transfer techniques, included administration of genes for the expression of angiogenesis growth factors into blood vessels were found to be both feasible and efficient
In vitro and experimental in vivo investigations, utilizing gene therapy techniques for pro-angiogenesis purposes, demonstrated increased formation of collateral blood vessels and functional improvement of ischemia (Refs. 8,9). In animal models of ischemia, there is evidence that proves that administration of angiogenetic growth factors (either in the strategy of gene transfer for the purpose of gene expression, or by direct administration of the recombinant protein to the tissue) can augment profusion through neovascularization. The best-studied model is with vascular endothelial growth factor (VEGF) and fϊbroblast growth factor (FGF). However, delivery of a single angiogenesis factor such as FGF or VGF is probably not sufficient to produce a fully organized and functional vasculator, since this process requires co-activation and interaction of a number of angiogenetic factors simultaneously (Ref. 6).
The H19 gene is one of the few genes known to be imprinted in humans. At the very beginning of embryogenesis, H19 is expressed from both chromosomal alleles, while shortly afterwards silencing of parenteral alert occurs, and only the maternally inherited allele is transcribed. HI 9 is abundantly expressed during embryogenesis and was first identified as a gene that was coordinately regulated with alpha-setaprotein in liver by the transacting locus RAF.
While transcription of the H19 RNA occurs in a number of different embryonic tissues throughout fetal life and placental development, H19 expression is down regulated postnatally. There have been reports that H19 is activated postnatally in cancer cells that
led to the speculation that H19 is an oncofetal RNA that can be used as possible human markers.
U.S. Patent 5,955,273 refers to H19 used as a tumor marker for the identification of bladder carcinoma and US 6,087,164 and 6,306,833 concern the use of the H19 promoter to express heterologous sequences in tumor cells, for the purpose of destruction of said cells in the course of cancer treatment.
Although H19 RNA is transcribed by the RNA Polymerase π, and is known to be spliced and polyadenylated, it does not appear that the H19 mRNA is translated into a protein. Instead, HI 9 RNA has been found to be associated with the 28 cytoplasmatic RNA, leading to the speculation that HI 9 RNA may function as an RNA component of the ribonucleoprotein. The actual physiological role of HI 9 has not been fully understood and elucidated to date, and has been hypothesized that it may be involved in the imprinting the IGS-2 gene, may be involved in tumor suppressor RNA, the effect of HI 9 as tumor suppressor being controversial, some evidence indicating the HI 9 in fact is over expressed in a large number of tumors.
SUMMARY OF THE INVENTION
The present invention is based on the surprising finding that a bladder carcinoma cell line, which endogenously does not express H19 RNA, showed a marked difference in gene-expression patterns when transfected with HI 9 sense, as compared with the gene- expression patterns of the same cell line, when transfected with the H19 antisense. In particular, the expression pattern with cells transfected with the H 19 sense, showed a marked increase in two unique groups of genes: one group that controls angiogenesis, and another group of genes which protects cells against ischemic stress.
These findings lead the way to the development of a novel method for regulating (increasing or decreasing) the expression of angiogenesis-controlling genes, by modulating (increasing or decreasing, respectively) the expression of H19.
By another aspect, these findings lead the way to the development of a novel method for regulating the expression of ischemic-stress induced genes by regulating the expression of HI 9 .
The method of the present invention has many utilities. By one, it may be used as a tool in basic scientific research, for finding out clusters of angiogenesis- confrolling genes or clusters of ischemic-stress induced genes which work in concert, and which expression can be regulated by modulating the expression of H19. This will allow exploring various scientific issues related to the regulation of expression of these angiogenesis controlling genes or ischemic-stress induced genes separately, and in combination, as well as aid in the research of the effect of various angiogenesis modulating factors or ischemic-stress induced factors on the expression of these genes.
The method may also be used for the production of various in vitro models of cells which over-, or under-express clusters of angiogenesis- controlling genes or of ischemic- stress induced genes, so as to study the effect and interaction of the these genes, as well as to the use of these cells for screening for agents, to be used as drugs, for the treatment of angiogenesis-related disorder (both for inhibition or stimulation of angiogenesis), or as agents used to treat ischemic-stress related disorders.
By another aspect, the method of the present invention, may be used for the production of genetically engineered animals, which over, or under express a group of angiogenesis- controlling genes, or ischemic -stress induced genes by constructing animals which over or under express the H19 gene, respectively, for the development of a model for research of drags affecting angiogenesis in general, and angiogenesis-related diseases and disorders in particular. In such animals the expression of the HI 9 sense or the H19 antisense should be under the control of an inducible promoter. It is also possible to construct genetically engineered animals as described above to study various aspects related to ischemia, as well as for the study of drags for the treatment of ischemic-stress related disorders.
By a most preferred aspect, the above method of regulating angiogenesis- controlling genes may be used for therapeutical purposes, i.e. to prevent, treat, or alleviate at least one of the undesired syndrome of diseases, disorders or pathological conditions which are manifested by non-normal (over- or under) angiogenesis.
By one aspect, termed the "pro-angiogenesis aspect" the therapeutical methods is used to increase the expression of angiogenesis-controlling genes, for the purpose of prevention, treatment or alleviation of diseases or conditions wherein a beneficial therapeutical affect may be evident by neovascularization.
Examples of such diseases are coronary artery diseases, peripheral artery diseases, endothelial vascular diseases, arteriosclerosis, various processes of wound and tissue healing such as healing of bone, tendon, endothelial lining (such as in ulcers in the stomach), for improving the success rates of cell transplantation techniques, as well as in reconstractive surgery to help re-estabhsh proper blood circulation to the reconstructed tissue.
By another aspect termed "anti-angiogenesis aspect." the method of the present invention may be used for the treatment, prevention or alleviation of diseases, conditions and disorders wherein a therapeutical beneficial affect is evident through the inhibition or the prevention of angiogenesis .
Examples of such diseases are cancer, aged-related macular degeneration (which are many times aggravated by normal neovascularization), diabetic retinopathy (which are also caused by non normal neovascularization), rheumatoid arthritis, psoriasis, obesity, hemagioma (AIDS related), kapossi's sarcoma arteriosclerosis, and restenosis.
Thus, the present invention concerns a method for regulating the expression of angiogenesis-controlling genes in cells which are involved in neo- vascularization, the method comprising:
Administering to the cells an effective amount of an H19 modulator in such a manner, and under such conditions, which enable change of the level of H19 expression, thereby regulating the expression of the angiogenesis-contiolling genes.
The term "regulating" in the context of the present invention concerns increase in the basal level expression of angiogenesis-controlling genes (in the "pro- angiogenetic" aspect of the invention), or decrease in the basal expression of the angiogenesis-contiolling genes (in the "anti-angiogenetic aspect" of the present invention), as well as increase or decrease in the inducible level of the HI 9 expression in response to external cues and in
particular change in the level of these genes in response to stress such as in response to hypoxia or ischemia.
The term "angiogenesis-controlling genes" refers to genes, which . expression product brings about, either directly or indirectly, to an increase in neovascularization process . This neovascularization may be due to growth of blood vessels, and/or due to the penetration of the growing blood vessels through the matrix, and/or due to the proliferation of endothelial cells which precedes both processes. The cells effected by the H19 modulator may also be cells that secrete angiogenesis-modulating agents (such a the stimulators and inhibitors mentioned in the Background of the Invention that effect angiogenesis. The effect may be the direct effect of the expression product of the gene, or may be the effect of another cellular component which is affected by the product of the gene or downstream in the pathway.
The product of the angiogenesis-controlling genes may bring about the angiogenesis effect by itself, but more commonly a number of such expression products from a number of angiogenesis-contiolling genes acting together, can bring about the desired angiogenesis effect. In fact, one of the advantages of the present invention resides in the fact that the H19 expression product can be a positive regulator (and an anti-H19 can be a negative regulator, in an analogous manner) of a set of genes that work in concert together. The activation of a number of angiogenesis controlling genes simultaneously by a single effector (H19 modulator), can induce normal neo-vascularization, which is a complicated process requiring the interaction of a number of genes. This effect may be better than previous attempts at neo-vascularization induction by the delivery of a single gene product such as FGF or VEGF, which were shown to induce abnormal unorganized vasculature (Ref. 6).
Examples of angiogenesis-controlling genes are: uPAR, IL-6, VEGF, HB-EGF, COX-2, TNFα, NF- B, sI-CAM-1, ReLa, c-SRC, HOOF (hypoxia inducible factor), angiostatin, endostatin, FGF-1, AFGF, ORK tyrosine kinase, tie tyrosine kinase, PDGF, IGF-1, integrine plasmic, endothelial-cell-specific molecule (ECSM), TGFβ, FGFα
(BFGF), EGF, HGF, HK, G-CSF, HGF, SF, IL-8, Leptin, Midkin, pleiotrophin (PTN) prolifern, TGFα, TGFβ, VPF
The term "cells are involved in neo-vascularization" in the context of the present invention refers preferably to endothehal cells, and more preferably to vascular endothehal cells, but this term also include other cells which constitute blood vessel walls, such as smooth muscle cells and mesenchymal]. This term also refer to cells that do not form directly the blood vessels but nevertheless effect the blood-vessel forming cells, for example, by secretion of angiogenetic stimulators or inhibitors ,helping penetration through extracellular matrix etc.].
In accordance with the pro-angiogenesis aspect of the invention, the expression of the angiogenesis-controlling genes is unregulated, by an HI 9 modulator that increases the level of H19 expression.
By another aspect, termed the "ischemic-stress" aspect, the present invention concerns a method for regulating ischemic-stress induced genes, and this may use for therapeutical purposes, i.e. to prevent, treat, or alleviate at least one undesired condition, disease or pathological condition, which can be improved by changing the response of the vitro ischemic stress.
By one aspect, termed the "ischemic-stress protection aspect", the therapeutical method is used to increase the expression of ischemic-stress induced genes, for prevention, treatment or alleviation of diseases or conditions, wherein a beneficial therapeutical effect may be evident by increasing the blood/ oxygen supply to the tissue. Examples of such diseases are in general neurodegenerative diseases wherein blood supply to a brain region is decreased, conditions of stroke or where the blood supply to neural cells is blocked or decreased, condition of blockage of blood supply to muscle, and in particular hard muscle causing heart damage, condition of peripheral ischemic conditions such as manifested in diabetes.
By another aspect termed the "ischemic-stress promoting aspect" the method of the present invention may be used for the treatment, prevention or alleviation of diseases, conditions and disorders, wherein a therapeutical beneficial effect is evident through
increase of the sensitivity of the tissue or cells to damage caused by ischemic stress. Examples of such diseases are in general cancer and other proliferative diseases.
Thus, the present invention concerns a method for regulating the expression of ischemic-stress induced genes in cells, the method comprising:
Administering to the cells an effective amount of H19 modulator in such a manner, and under such conditions that enable change of the level of H19 expression thereby regulating the expression of the ischemia-stress induced genes.
The term "regulating" in the context of the present invention concerns increase in the basal level of the ischemic-stress induced genes (in accordance with the ischemic- stress protection aspect of the invention), or decrease in the basal expression of ischemia- stress induced genes (in accordance with the ischemic-stress promoting aspect of the present invention), as well as increase or decrease in the inducible level of these genes in response to external cures, and in particular in response to change in the level of blood or oxygen in the region.
The term "ischemia-stress induced genes" refers to genes which expression is induced by ischemic stress and which expression product brings about, either directly or indirectly, a decrease in the damage caused to cells in general due to decrease, or lack of oxygen or blood supply to the region.
The term "H19" refers to the H19 mRNA, as depicted for example in SEQ ID NO: 1, or a fragment of this sequence, which fragment is of sufficient length, and of an appropriate sequence so as to cause the increase in the expression of at least one angiogenesis-controlling gene, or at least one ischemic-stress induced gene.
In accordance with the pro-angiogenesis aspect of the invention and with the ischemic-stress induced aspect of the invention, the positive modulator of H19 is any agent that results in increasing the amount of HI 9 mRNA, or a fragment of such mRNA, in the cell.
Examples of such positive modulators of HI 9 are the following: (i) A molecule which comprises the H 19 RNA, or a fragment of the H 19 RNA, such that the sequence of this fragment is sufficient to increase the
expression of at least one angiogenesis-controlling gene. The HI 9 modulator may be the HI 9 mRNA itself, present with a specific carrier enabling its penetration into the cells, such as liposomes, may be bound to specific agents which allow transport of sequences to the cells, or may be present in the form of naked nucleic acid sequence. .
(ii) By a preferred alternative, the positive modulator of H19 is an expression vector that comprises a sequence coding for the mRNA of (i) above, i.e., either coding for the complete sequence of the HI 9 RNA, or coding for a fragment of the HI 9 RNA, the fragment having a sequence sufficient to increase the expression of at least one angiogenesis-contiolling gene, or at least one ischemic-stress induced gene.
The sequence coding for H19 may be present in the form of naked DNA, or may be present in synthetic vectors such as liposomes; or viral vectors, adenoviru's, adeno- associated virus, lentiviruses, refroviruses; alternatively, it may be present in cell based delivery systems (Refs. 6 and 7).
The vectors may be aάrninistered to the body by any mode known in the art such as by direct injection, systemic delivery, by use of catheter mediated local delivery directed to the site (for example, intracoronary, intramyocardial) (Ref. 7)..
In accordance with the anti-angiogenetic aspect of the invention or the ischemic- stress induced aspect of the invention, the method is used to decrease the expression of at least one angiogenesis-controlling gene, or decrease the expression of at least one ischemic-stress induced gene, by the use of a negative HI 9 modulator which causes a decrease in the level of H19 expression.
Examples of negative modulators of H19 expression are selected from: (i) A nucleic acid sequence (composed of RNTPs, DNTPs or a combination of both) which is complementary to the H19 mRNA, or complementary to at least a fragment of the HI 9 RNA, so that hybridization between the HI 9 mRNA and said nucleic acid sequence will result in the neutralization of the H19 mRNA so as to eliminate its activity As the HI 9 mRNA is not
translated to a protein, the neutralization may be by several mechanisms, such as by inhibition of the maturation process of the H19 hnRNA to mRNA; by inhibition of the transport of the RNA from the nucleus to the cytoplasm; by production of a double-strand RNA construct which is more liable to RNAas digestion (such as RNase H) digestion; or by production of a double-strand RNA construct that interferes with the interaction of the native H19 with the cellular components which it naturally effects .
(ii) The negative modulator may also be a nucleic acid sequence which is complementary to the HI 9 gene, or complementary to at least a fragment of the HI 9 gene, so that hybridization between the H19 gene and said nucleic acid sequence (forming a triple helix) result in the decrease of transcription from the H19 gene, and hence results in decrease in the amount of the H19 mRNA product.
(hi) Use of double stranded short RNA constructs knows as "interfering RNA "
(iRNA). (iv) A further option is a catalytic nucleic acid sequence (ribozyme) which can inactivate in a specific manner the HI 9 mRNA. This typically is achieved by specific hybridization of the ribozyme with the H19 mRNA and cleaving or splicing of the sequence thus causing its neutralization. An example of such a ribozyme is what is termed "angiozyme" which is a specific ribozyme targeting angiogenesis-RNA molecule so that it cleaves and digests the mRNA of VEGF1 (Clinical Trial Partnership between Chiron and Company Ribozymes). (v) Negative modulator of the HI 9 may also be a nucleic acid sequence
(preferably a DNA) coding for any one of (a), (b), (c) or (d) above. The nucleic acid sequences, for example those of (a) to (e) may be introduced as naked nucleic acid molecules as explained above, or in the form of synthetic vectors such as liposomes; in viral vector, adenovims, adeno-associated virus, lentiviruses, retioviruses, as well as be present in cell based delivery systems (Refs. 6 and 7).
The expression vector, as in (iv), may also be introduced as explained above in accordance with the pro-angiogenetic aspect of the present invention.
EXPERIMENTAL PROCEDURES
1. Cell Culture
Human bladder carcinoma cell, line T24P was obtained from the American type culture collection, USA. TA11 (stably transfected with antisense H19 RNA), and TA31 (stably transfected with full length HI 9 RNA) were obtained as previously described (Ref
1.)
The cells were grown as previously described. (Ref. 2). \
2. Gene constructs and transfection
H19 sense and antisense expression vectors were constructed as described before. A total of (0.6 ) cells plated in 30mm dishes were transfected with 7 μg of either plasmid (i.e. containing the H19 antisense to produce TAll, or containing the full length H19 sense to produce the TA31) using the calcium phosphate precipitation method. The cells were treated with 0.5-1 mg/ml G418 (Geneticin) 24 h after transfection to initiate selection.
3. Differential Hybridization of Atlas Human cDNA Expression Arrays
Atlas Human cDNA expression array membranes were purchased from Clontech (palo Alto, CA). Each membrane contained the cDNA from 588 known genes and 9 housekeeping genes. Simultaneous hybridization of the membranes with complex cDNA probes prepared from two different poly A+ RNA population isolated from TAll and TA31 (lug each) by reverse transcription in the presence of [alpha-32P] dATP allowed direct comparison of the expression level off all indicated cDNAs on the array. Hybridization was done overnight using ExpressHyp™ hybridization solution, followed by gh-stringency wash. The hybridization pattern was then analyzed by autoradiography, qualified by scanning with power look II scanner and analyzed wuth Image-Pro Plus software (Media Cybernetics, USA) with the algorithm for color transformation and digital
image analysis was developed in the lab. The relative expression level of any given gene was assessed by comparing the signal obtained with the probes prepared from either the TA31 ot TAll RNA.
4. Selection of specific genes for individual RT-PCR analysis
A few genes that showed variation between the two different cell lines were selected for further RT-PCR analysis to confirm the results obtained by hybridization to the human Atlas array. The sequense of the primers for the selected genes were obtained from Clontech. These genes were : Sterol regulatory element-binding protein, Macrophage inflammatory protein-2-alpha, RNA polymerase II elongation factor SHI, and Interleukin- 6. Conditions were optimized in order to ran the RT-PCR for all the four genes at the same number of cycles. From the same poly A+ RNA used in the hybridization protocol, 0.1 ug from each sample was used in the RT-PCR reaction in a 40 ul final volume. At the end of PCR, 10 ul were loaded on 2.5% agarose gel and stained with Syber-green. The images of the scanned agarose gels were analyzed using Image-Pro Plus software to quantify the specific band of the indicated gene -specific RT-PCR product. (Fig 6)
5. RT-PCR Analysis for p57 kip2 and PCNA
A p57 Kip2 gene is responsible for proliferation of cells and proliferation is the first step in the angiogenesis process.
Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed to synthesize the cDNA from T24P and its derivatives TAll and TA31, in the presence and absence of serum. Cells were grown in the first day in the presence of serum and when they were at 40% confluency media were changed and media with 0.1% FCS were added. Control experiment was run simultaneously in the presence of 10% FCS.
After 72(hr) of serum starvation total RNA was extracted by RNA-STAT 60 kit following product instructions and the samples where treated by DNAse to exclude contamination of genomic DNA.
For (RT-PCR) reaction, the synthesis of cDNA was performed using the p(dT)15 primer (Boehringer, Mannheim, Germany), to initiate reverse transcription of 5ug total
RNA with 400 units of Reverse Transcriptase (Gibco BRL), according to the kit instructions. 2ul of the cDNA was used as a template for a PCR reaction using Taq polymerase (Takara) to amplify p57Kip2 transcript using PCR primers 5-CGATCAAGAAGCTGTCCGGGCCTC-3 (upstream) and 5-CCGCCGGTTGCTGCTACATGAACG-3 (downstream) In a 25 ul reaction volume. The PCR program was run as follows: Initial denaturation for 5 rnin at 94.0,followed by denaturation for 30S at 94.0, annealing for 30S at 58.0, elongation for 30S at 72.0, and this was run for 29 cycles followed by 3 min elongation at 72.0. 3% DMSO was added to the reaction mixture.
PCNA transcript amplification was done using the following PCR primers: 5-GACGGTGTTGGAGGCACTGAAGGAC-3 (upstream) and 5-GGTGCTTCAAATACTAGCGCCAAGG-3 (downstream). The PCR program was run as follows: Initial denaturation for 5min at 94.0, followed by denaturation for 15S at 98.0,annealing for 40S at 60.0, elongation for 45S at 72.0,and this was run for 30 cycles followed by 5rnin elongation at 72.0. 5% DMSO was added to the reaction mixture. The PCR product was separated by electrophoresis on 2.5% agarose, and detected by ethidrum bromide dye.
6. Cell Proliferation assay
A. Alamar Blue Assay:
Effect of serum starvation on the growth of T24p ,TA11, and TA3 l,were analyzed using Alamar blue assay.
Alamar blue is ready to use dye solution which shifts from blue to pink depending on the amount of oxido-reduction reactions in the cell system where its reduction is proportional to the number of viable cells and hence it is applied to detect proliferation of cells.
The cells (T24p,TAll, and TA31) were cultured in triplicates in 24-well (Nunclon 24 flat bottom) at an initial density of 3*10 ((4)) cells /well in 1ml medium. The cells were given to grow in a medium containing 10% FCS for 24hr, afterwards, part of the wells
were subjected to medium deficient in serum (0.1% FCS) for 72 hr and the other part continue to receive medium containing 10% FCS as a control.
Alamar blue (TM) (Biosource,CA) was added 50ul/well immediately after placing media and the plate was assayed 2 hr later for fluorescence emission using fluorometer at wavelengths of 530nm exitation/590 nm emission. B. Brdl F incorporation:
A second method was used to asses the difference in proliferative capacity in T24p and its derivatives TA11 and TA31 in response to serum depriviation, by measuring BrdU incorporation. The scheme outlined above was followed here except that the cells were plated in 96-well (Nunclon 96 flat bottom at an initial density of 3*10((3)) cells/well in quadrable.
After 72br of serum deprivation media was changed and the cells were incubated with BrdU for 2hr according to kit instructions (cell proliferation Elisa, BrdU colorimetric) (Roche).
Absorbance was measured using Elisa reader at 370nm (reference wavelength 490nm)
Example 1 Expression of angiogenesis-controlling genes in +H19 and -H19 cells
Human bladder carcinoma cell line T24P, which does not have endogenous H19 mRNA, were transfected with an expression vector having the H19 transcript (SEQ ID NO: 1) in the sense direction (to produce the TA31 cells) or the H19 antisense transcript (SEQ ID NO: 2) (to produce TA11 cells). The heterologous sense or anti-sense sequence was placed under the CMV promoter.
The mRNA of the TA31 and TAll cells was analyzed in a cDNA artery microarray
The results showing the difference of expression of angiogenesis-contiolling genes are shown in Table 1.
Table 1: Upregulated genes in TAll and TA31 cells (only genes showing at least a twofold activation are described)
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