Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-03
2001-07-03
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007300, C435S091200, C436S064000
Reexamination Certificate
active
06255055
ABSTRACT:
BACKGROUND OF THE INVENTION
The c-myc protein is a member of the helix-loop-helix/leucine zipper (HLH/LZ)
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family of transcription factors that forms heterodimers with Max (1-3). In general, trans-activating Myc:Max heterodimers are found in proliferating cells, while trans-repressing Mad:Max heterodimers are found in differentiated cells. The c-myc protein level influences cell proliferation, differentiation, and neoplastic transformation, presumably by affecting the balance between Myc:Max and Mad:Max heterodimers (4). When c-myc protein is overexpressed or is induced at inappropriate times, this balance is perturbed, and cell proliferation and differentiation are disrupted. For example, c-myc overexpression prevents or delays cell differentiation (5, 6). It also blocks serum-starved cells from entering the G
o
phase of the cell cycle and instead induces them to undergo apoptosis (7). c-myc overexpression is also implicated in tumor formation in experimental animals and in human patients with Burkitt's lymphoma (8, 9). These and other deleterious consequences of aberrant c-myc expression highlight the importance of understanding all aspects of c-myc gene regulation.
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The abbreviations used herein are: HLH/LZ, helix-loop-helix/leucine zipper; AURE, AU-rich element; UTR, untranslated region; CRD, coding region determinant; CRD-BP, coding region determinant-binding protein; DTT, dithiothreitol; EGTA, ethylene glycol bis(2 aminoethyl ether)-N,N′ (tetraacetic acid); PMSF, phenylmethyl-sulfonylflouride; S130, post-polysomal supernatant; SDS, sodium dodecyl sulfate; RSW, ribosomal salt wash; PCR, polymerase chain reaction; bp, base pairs; EST, Expressed Sequence Tags; RACE, rapid amplification of cDNA ends; BAC, Bacterial Artificial chromosome; GCG, Genetics Computer Group; IP, immunoprecipitation; mRNP, messenger ribonucleoprotein; hnRNPK, heterogeneous nuclear ribonucleoprotein K; HRP, horseradish peroxidase; HSP-90, heat shock protein-90; MOPS, morpholinepropanesulfonic acid; KH, K homology; ORF, open reading frame; FMR, familial mental retardation; FMRP, FMR RNA-binding protein; hKOC, human KH domain protein overexpressed in human cancer; PAG, polyacrylamide gel; PAGE, polyacrylamide gel electrophoresis; ECL, enhanced chemiluminescent.
The c-myc protein is regulated by phosphorylation, protein:protein interactions, and changes in its half-life (10-12). c-myc mRNA levels are regulated transcriptionally and post-transcriptionally, and changes in c-myc mRNA stability can result in large fluctuations in c-myc protein levels. The c-myc mRNA half-life is normally only 10 to 20 minutes but can be prolonged 3- to 6-fold when necessary. For example, c-myc mRNA is relatively stable in replicating fetal rodent hepatocytes, which produce abundant c-myc mRNA. It is far less stable in non-growing adult hepatocytes, which contain little or no c-myc mRNA (13, 14). However, it is up-regulated and stabilized several-fold when adult hepatocytes replicate following partial hepatectomy (15, 16).
Two cis-acting sequence elements in c-myc mRNA contribute to its intrinsic instability and perhaps also to its post-transcriptional regulation: an AU-rich element (AURE) in the 3′-untranslated region (3′-UTR) and a 180 nucleotide coding region determinant (CRD). The CRD encodes part of the HLH/LZ domain and is located at the 3′ terminus of the mRNA coding region. Four observations indicate how the c-myc CRD functions independently of the AURE to affect c-myc mRNA expression. (i) c-myc mRNA lacking its CRD is more stable than wild-type c-myc mRNA (17-20). (ii) The CRD is required for the post-transcriptional down-regulation of c-myc mRNA that occurs when cultured myoblasts fuse to form myotubes (20, 21). (iii) Inserting the c-myc CRD in frame within the coding region of &bgr;-globin mRNA destabilizes the normally very stable &bgr;-globin mRNA (22). (iv) The c-myc CRD is necessary for up- and down-regulating c-myc mRNA levels in transgenic mice undergoing liver regeneration following partial hepatectomy (13, 15, 16, 23-25). In summary, the c-myc CRD influences c-myc mRNA stability in animals and in cultured cells.
We have investigated c-myc mRNA stability and the function of the CRD using a cell-free mRNA decay system that includes polysomes from cultured cells. The polysomes contain both the substrates (mRNAs) for decay and at least some of the enzymes and co-factors that affect mRNA stability. Polysomes are incubated for different times in an appropriate buffer system, and the decay rates of polysomal mRNAs such as c-myc are monitored by hybridization assays. This system reflects many aspects of mRNA decay in intact cells (26-29). For example, mRNAs that are unstable in cells are also relatively unstable in vitro; mRNAs that are stable in cells are stable in vitro (26). In standard reactions, the polysome-associated c-myc mRNA was degraded rapidly in a 3′ to 5′ direction, perhaps by an exonuclease (29). An alternative decay pathway became activated when the reactions were supplemented with a 180 nucleotide sense strand competitor RNA corresponding to the c-myc CRD. This CRD RNA induced endonucleolytic cleavage within the c-miyc CRD, resulting in an 8-fold destabilization of c-myc mRNA (30). These effects seemed to be specific for c-myc. Other competitor RNAs did not destabilize c-myc mRNA, and c-myc CRD competitor RNA did not destabilize other mRNAs tested.
Based on these observations, we hypothesized that a protein was bound to the c-myc CRD. We further suggested that this protein shielded the CRD from endonuclease attack, that the CRD competitor RNA titrated the protein off of the mRNA, and that the unprotected c-myc CRD was then attacked by an endonuclease. Consistent with this model, we detected a protein that binds strongly in vitro to a c-myc CRD
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P-RNA probe (30). This protein, the c-myc coding region determinant-binding protein (CRD-BP), was subsequently purified to homogeneity (31). We then found that the CRD-BP is developmentally regulated, being expressed in fetal and neonatal rats but not in adult animals (32).
SUMMARY OF THE INVENTION
In the Examples below, we report the cloning of the mouse CRD-BP cDNA, a novel member of an RNA-binding protein family. We also show that the CRD-BP can bind to ribosomes in vitro and that most of the CRD-BP in cell extracts is located in the cytoplasm and is associated with polysomes and ribosomes. These observations are consistent with a role for the CRD-BP in shielding polysomal c-myc mRNA from endonucleolytic attack, which means that the CRD-BP helps to preserve c-myc mRNA and allows it to be used to make c-MYC protein. We believe that blocking CRD-BP expression might result in the very rapid destruction of c-myc mRNA and subsequent depletion of c-MYC protein from the cell.
We have also shown that the CRD-BP is abundantly expressed in cancer cell lines grown in the laboratory as well as in fetal tissues from rodents (32). In contrast, the CRD-BP is undetectable in tissues from adult rodents (32). We believe that these latter observations may be consistent with the idea that the CRD-BP is an oncofetal protein—that is, a protein that is expressed in the fetus and in cancer cells in post-natal life but is not expressed in normal (non-cancerous) tissues in post-natal life. If so, then the CRD-BP should be present in cancer tissues but not in normal tissues in post-natal life.
Specific, restricted expression of the CRD-BP in cancerous tissues could mean that the CRD-BP is a potential diagnostic/prognostic marker for human cancer. Moreover, since the CRD-BP seems to protect c-myc mRNA from being destroyed rapidly, and since c-MYC protein is essential for cell growth, then eliminating the CRD-BP from cancer cells could lead to the cessation of their growth or even to their death.
The present invention is a method of diagnosing the presence or absence of cancer in a human patient comprising the steps of examining patient tissue for the CRD-BP expression levels and comparing that expression level with a control or exami
Caputa Anthony C.
Hunt Jennifer
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
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