Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2000-11-03
2002-08-27
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S039000
Reexamination Certificate
active
06440658
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a biological assay method. Particularly, it pertains to a method for determining the effect of Product R on adenovirus infection of Hela cells.
2. Description of the Related Art
Product R is an antiviral agent useful for treating a wide range of viral infections, such as infections of human immunodeficiency virus (HIV), herpes simplex virus, adenovirus. It has become known that Product R is effective in stimulating the production of chemokines, including interferon-gamma, interleukin-6 and interleukin-1. Product R is described in detail in U.S. patent application Ser. No. 09/344,095, which is incorporated herein by reference in its entirety. However, the mechanism of Product R in treating human viral infections is yet to be fully understood. Improved methods for detecting and measuring Product R's existing or potential biological activities are thus desired. To applicant's knowledge, no one has heretofore taught or suggested any assay directing to measuring Product R's effect on adenovirus infection of Hela cells.
Significant progress has been made in unraveling the details of the molecular circuits that regulate the cell cycle engine, as well as of the surveillance mechanisms (checkpoints) that ensure that chromosome duplication and segregation take place only when appropriate. In mammalian cells, both DNA synthesis (passage through G
1
into G
2
phase) and re-entry into the cell cycle from G
0
depend on external growth factors and other agents that stimulate cell growth and division (mitogens). Late in G
1
cells responding to such external cues become committed to enter S phase, divide and complete the cell cycle. During this time they are refractory to extracellular signals that regulate growth. Cells that have entered this state are said to have passed the G
1
restriction point. The genomes of several DNA viruses encode proteins that are mitogenic because they subvert the normal mechanisms of restriction point control. Indeed, the functions of critical components of these cellular regulatory mechanisms has initially been deduced through the activity of such viral proteins.
The most unexpected result to emerge from these genetic studies is the difference between the 5V40 and polyomavirus early proteins needed for transformation. SV40LT is essential for induction of transformation, with sT being required for the expression of specific phenotypes in certain cell types. In contrast, polyomavirus LT is not sufficient for transformation, nor are the sequences encoding its C-terminal segment necessary. Rather, polyomavirus middle T antigen, an early protein that has no counterpart in the 5V40 genome, is necessary both to establish and maintain the transformed state.
Although the human pathogens HILV-1 and HIV-1 both have complex genomes that encode regulatory proteins, they belong to two distinct groups. HIV-1 is a member of the lentivirus group. Although HIV-1 is not known to transform cells that it infects, a relatively high incidence of an otherwise rare cancer, called Kaposi's sarcoma, is associated with AIDS. This type of tumors is thought to be associated with expression of the HIV regulatory protein Tat, an idea supported by the finding that mice transgenic for HIV Tat develop a disease analogous to Kaposi's sarcoma. The Tat protein has an RGD domain like that found on extracellular matrix proteins and thus may stimulate integrins on epithelial cells, causing inappropriate proliferation. HIV-1 encodes another protein, called Vpr, which prevents proliferation of infected cells by arresting them in the G
2
phase of the cell cycle. (1) This Vpr-mediated cell-cycle arrest has also been observed in several highly divergent simian immunodeficiency viruses, suggesting an important role for this protein in the virus life cycle. The expression of the viral genome is optimal in the G2 phase of the cell cycle, and Vpr increases viral production by delaying cells at the point of the cell cycle where the long terminal repeat (LTR) is more active.
Adenovirus is an ideal model for studying the interaction between cellular and viral genes in gene regulation. The cellular DNA-binding protein, E2F, was identified originally by its ability to bind to a specific recognition sequence in adenovirus E2 promoter. In addition, the viral protein, E1A has been shown to induce E2F-mediated DNA binding and transcriptional activities by releasing free E2F from inactive protein complexes. The significance of these findings was limited until it was observed that promoters of many cellular genes contain similar E2F-binding sites and that E2F is one of the important cellular transcription factors in regulating expression of some of these genes. Many of these genes are involved in cell cycle progression, particularly in DNA synthesis. Furthermore, several key regulators of the cell cycle, including the retinoblastoma protein (Rb) and related proteins p107 and p130, were found to form complexes with E2F, indicating the potential role of E2F in cell cycle progression. By its ability to bind to the Rb protein, E2F-1 was the first gene product identified among a family of E2F transcription factors. As an authentic transcription factor, E2F-1 contains a specific DNA-binding domain and a potent transactivation region. E2F-1 can form heterodimers with another E2F-like protein, DP-1, and have a synergistic effect on its transcription activity. The Rb-binding domain of E2F-1 overlaps its transcriptional activation region, suggesting a possible mechanism for Rb to regulate E2F-1 transcriptional activity. Indeed it has been shown that Rb suppresses transcriptional activation mediated by E2F-1 through the direct interaction between the two molecules. The inhibitory effects of Rb can be disrupted by its direct interaction with viral oncoproteins, such as E1A, an effect similar to that achieved by mutation or phosphorylation of the Rb protein. A noteworthy observation made during the original characterization of E2F-1 was that expression of this protein is cell cycle dependent, with a peak at the G
1
/s boundary. This finding is consistent with the hypothesis that E2F functions primarily at this period of time in the cell cycle and that E2F-1 mediated transcriptional activation may be one of the rate-limiting steps in cell proliferation. Indeed, deregulated expression of E2F-1 in Rat-2 fibroblasts was found to induce premature entry into S-phase, subsequently leading to apoptotic cell death.
Cyclin B is first synthesized during S phase, accumulates in complexes with p34
cdc2
as cells approach the G
2
to M transition, and is abruptly degraded during mitosis. Phosphorylation of p34
cdc2
on threonine-161 may stabilize its binding to cyclin B and is required for the subsequent activation of the enzyme. Other phosphorylations at threonine14 and tyrosine-15 within the p34
cdc2
ATP-binding site maintain the kinase in an inactive form throughout S and G
2
. Removal of the inhibitory phosphates from cyclin B-associated p34
cdc2
at the G
2
/M transition activates the p34
cdc2
kinase and triggers entry into mitosis. Conversely, exit from mitosis depends upon the abrupt ubiquitin-mediated degradation of cyclin B during anaphase, resulting in the release of p34
cdc2
as an inactive monomer. Checkpoint controls impinging upon the kinases and phosphatases that regulate p34
cdc2
activity ensure that S phase ends before mitosis begins.(1)
Progression of eukaryotic cells through the cell cycle is governed by the sequential formation, activation, and subsequent inactivation of a series of cyclin-dependent kinase (Cdk) complexes. The mechanisms underlying the expression of cyclins and the activation of the different cyclin-Cdk complexes needed for progression through the successive cell cycle transitions are now fairly well understood. In addition to positive regulation by the activation of cyclin-Cdk complexes, negative regulation of the cell cycle occurs at checkpoints, which are the transitions where feedback mecha
Hirschman Shalom Z.
Huang Wenlin
Advanced Viral Research
Cohen & Pontani, Lieberman & Pavane
Park Hankyel T.
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