Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-06-04
2004-09-21
Helms, Larry R. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007920
Reexamination Certificate
active
06794151
ABSTRACT:
BACKGROUND OF THE INVENTION
The c-myc protein is a member of the helix-loop-helix/leucine zipper (HLH/LZ)
1
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 apodtosis (
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. ≠
1
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-myc 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
32
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 expres
Helms Larry R.
Yu Misook
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