Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-02-18
2002-04-09
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023500, C536S024500
Reexamination Certificate
active
06368790
ABSTRACT:
BACKGROUND OF THE INVENTION
Differentiation in many cell lineages has been established to be a multistep process. This is perhaps best illustrated by analysis of the differentiation of 3T3T mesenchymal stem cells into adipocytes (1). Undifferentiated 3T3T cells first arrest their proliferation in the G
1
phase of the cell cycle at a distinct state prior to differentiation. Associated with this process, expression of the PPAR&agr;2 lineage specific transcription factor is induced (2). Thereafter, the C/EBP family of transcription factors is expressed to induce a series of adipocyte differentiation genes that include 422, GDPH, lipoprotein lipase and adipsin (3, 4, 5). The resultant adipocytes are nonterminally differentiated because they can be induced to reinitiate proliferation and reenter the cell cycle. Adipocytes at the nonterminal state of differentiation can however be induced to terminally differentiate by exposure to aproliferin and thereby irreversibly lose their growth factor responsiveness (6). When terminal adipocyte differentiation occurs a marked repression in the expression of P2P proteins is evident (7).
P2Ps, i.e. proliferation potential proteins, comprise a group of highly basic 35-40 kDa nuclear proteins that can bind to RNA and are associated with hnRNP particles as determined by sucrose gradient sedimentation of nuclear components (7). In this application, references to a singular P2P protein encompass the plural P2P “proteins”, and vice versa. Antibodies prepared against core hnRNPs recognize P2Ps and 2D gel electrophoresis established that P2Ps are members of the A/B class of hnRNP proteins which are involved in RNA processing (7,9).
Terminal differentiation has also recently been demonstrated to require the expression of the tumor suppressor protein Rb1 (10). In studies using myoblasts derived from native animals that express Rb1 and myoblasts from transgenic animals that lack Rb1, it was established that cells lacking Rb1 cannot terminally differentiate. Instead, they are blocked at a state of nonterminal differentiation. These observations suggest that the function of Rb1 as a tumor suppressor gene product may be related to its role in the control of terminal differentiation. This possibility is supported by data showing that the Wilms' tumor suppressor gene product WT1 is also involved in the terminal differentiation of renal blastema cells during neonatal development (11). Recent reports also show that the Rb1 and WT1 proteins can be localized in the nucleus to sites of RNA processing suggesting that tumor suppressor mechanisms may be mediated by regulating the processing of specific mRNAs (12,13).
SUMMARY OF THE INVENTION
The invention is a novel P2P cDNA, the protein/proteins encoded by the P2P cDNA, monoclonal antibodies against P2P protein, a diagnostic method which involves detection of the DNA, an RNA transcribed by the DNA, or the protein, P2P antisense reagents derived from the P2P cDNA, and a method for gene therapy using these reagents.
The invention is partly based on evidence that the irreversible loss of proliferative potential is associated with repression in the expression of hnRNP-associated proteins that are involved in RNA processing which was published in 1989 (7). It was specifically demonstrated that the terminal differentiation of 3T3T adipocytes correlates with a markedly decreased expression of a set of proteins designated P2P, i.e. proliferation potential proteins, P2Ps were shown to have a pI of greater than 9.0, to range in size from 35 to 40 kDa and to localize to nuclear hnRNP particles as determined using sucrose gradient sedimentation methods. Additional studies established that P2Ps are recognized by the FA12 monoclonal antibody that detects purified core hnRNP proteins. The results of 2D gel electrophoresis further established that P2Ps are type A/B hnRNP proteins. P2Ps also share an epitope in common with hsp90 as determined by use of the AC88 monoclonal antibody even though P2Ps are not heat shock proteins. Subsequently, the terminal differentiation of human keratinocytes was also shown to be associated with a marked decrease in P2P expression (7) and P2P expression was shown to markedly decrease in association with the senescence of normal human cells (8). In contrast, it was shown that P2P expression is not repressed when malignant cells differentiate (7).
A description is provided of the cloning and characterization of the P2P cDNA. The result of this effort defined a 5173 base pair cDNA, shown in
FIG. 6
, containing a 4214 base pair open reading frame encoding a 156.9 kDa protein. The deduced amino acid sequence of the P2P open reading frame shows a highly basic protein, i.e, pI 9.6, as predicted. Probes to the P2P cDNA detect a single 8 kb mRNA in urine kidney, liver, testes, lung and other tissues and in growing murine 3T3T mesenchymal stem cells. In contrast, P2P mRNA expression is markedly decreased when 3T3T cells undergo the terminal step in the process of adipocyte differentiation. However, P2P mRNA expression is not repressed in nonterminally differentiated adipocytes suggesting that regulation of P2P expression is associated specifically with terminal differentiation.
To establish that the P2P cDNA encodes the P2P subset of hnRNPs, a series of monoclonal antibodies was prepared to a P2P cDNA-derived fusion protein, one of which is designated C130. Other monoclonal antibodies prepared from the P2P cDNA-derived fusion protein are designated C50, C147, and C167. The C130 antibody was shown to detect native 35 to 40 kDa P2Ps and other higher molecular weight products of the P2P cDNA, including a low abundance −160 kDa protein. This protein is recognized by C130 when nuclear extracts are fractionated by single stranded DNA affinity chromatography. This high molecular weight protein is thought to represent the intact product of the P2P cDNA which then appears to be processed into lower molecular weight P2Ps. Since monoclonal antibodies C130 and AC88 both detect P2Ps which are proven hnRNP's of the A/B subtype, this data provides support for the conclusion that the P2P cDNA encodes hnRNP-associated proteins. Data also show that a P2P antisense oligonucleotide selectively represses 30-40 kDa P2P expression.
Studies were next performed to determine if Rb1 might interact with P2P cDNA products. This possibility was suggested by data showing that Rb1 is involved in terminal differentiation and in other growth control mechanisms. Evidence that the P2P cDNA does encode a Rb1 binding protein was obtained by analysis of the Rb1 binding characteristic of GST-P2P fusion proteins. GST-P2P (753 to 909) was specifically shown to bind Rb1. The fact that Rb1 binding to this fusion protein is specifically competed by E1a suggest that the binding occurs to the Rb1 “pocket” domain (28,29). These data are consistent with data concerning the RBQ1 cDNA which was selected based on its ability to bind Rb1 and the fact that the RBQ1 cDNA shows significant homology to the 5′ portion of the P2P cDNA (23).
The deduced P2P cDNA product also contains additional interesting domains. The first of these is a cell division sequence motif [CDSM] that has been proposed to be characteristic of proteins involved in the regulation of cell division (27). Examples of proteins that contain this motif include cdc 25, c-myc and several viral proteins including E1a, E7 and SV40 large T antigen. The presence of the CDSM in the P2P cDNA product is consistent with the evidence showing that P2Ps are involved in regulating a cells proliferative potential. Another distinct domain encoded by the 5′ portion of the P2P cDNA is a cysteine-rich region that is related to “ring” zinc fingers (30). These zinc finger domains are thought to define protein conformation characteristics that are involved in nucleic acid binding and protein-protein interactions. These attributes are compatible with the fact the P2Ps are known to bind to single stranded DNA and to associate with other hnRNP proteins (31).
These data together suggest
Adler Benjamin Aaron
Brusca John S.
University of Tennessee Research Corporation
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