Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-11-21
2004-01-27
Crouch, Deborah (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S320100, C435S325000, C424S093210
Reexamination Certificate
active
06683059
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methodology and associated genetic constructs for the suppression of oncogenic transformation, tumorigenesis and metastasis.
BACKGROUND
An extensive body of research exists to support the involvement of a multistep process in the conversion of normal cells to the tumorigenic phenotype (see, e.g., Land et al., 1983). Molecular models supporting this hypothesis were first provided by studies on two DNA tumor viruses, adenovirus and polyomavirus. In the case of adenovirus, it was found that transformation of primary cells required the expression of both the early region 1A (E1A) and 1B (E1B) genes (Houweling et al., 1980). It was later found that the E1A gene products could cooperate with middle T antigen or with activated H-ras gene to transform primary cells (Ruley, 1985). In addition, during the last decade, a number of human malignancies have been discovered to be correlated with the presence and expression of “oncogenes” in the human genome. More than twenty different oncogenes have now been implicated in tumorigenesis, and are thought to play a direct role in human cancer (Weinberg, 1985). Many of these oncogenes apparently evolve through mutagenesis of a normal cellular counterpart, termed a “proto-oncogene”, which leads to either an altered expression or activity of the expression product. There is considerable data linking proto-oncogenes to cell growth, including their expression in response to certain proliferation signals (see, e.g., Campisi et al., 1983) and expression during embryonic development (Muller et al., 1982). Moreover, a number of the proto-oncogenes are related to either a growth factor or a growth factor receptor. These observations suggested the involvement of multiple functions in the transformation process, and that various oncogenes may express similar functions on a cellular level.
The adenovirus E1A gene codes for several related proteins to which a number of interesting properties have been attributed. In addition to its ability to complement a second oncogene in transformation, a closely related function allows E1A to immortalize primary cells (Ruley, 1985). For example, introduction of E1A gene products into primary cells has been shown to provide these cells with an unlimited proliferative capacity when cultured in the presence of serum. Another interesting action of E1A function is so-called “trans-activation”, wherein E1A gene products stimulate transcription from a variety of viral and cellular promoters, including the adenovirus early and major late promoter as well as other promoters. However, trans-activation is not universal for all promoters. In some instances, E1A causes a decrease in transcription from cellular promoters that are linked to enhancer elements (Haley et al., 1984). It has been shown that exogenously added E1A gene can reduce the metastatic potential of ras-transformed rat embryo fibroblast cells by activating the cellular NM23 gene that is associated with a lower metastatic potential (Pozzatti et al., 1988; Wallich et al., 1985).
In the case of Adenovirus 5, the E1A gene products are referred to as the 13S and 12S products, in reference to the sedimentation value of two mRNAs produced by the gene. These two mRNAs arise through differential splicing of a common precursor, and code for related proteins of 289 and 243 amino acids, respectively. The proteins differ internally by 46 amino acids that are unique to the 13S protein. A number of E1A protein species can be resolved by PAGE analysis, and presumably arise as a result of extensive post-translational modification of the primary translation products (Harlow et al., 1985).
Another viral oncoprotein, the SV 40 large T antigen (LT) shares structural and functional homology to E1A and c-myc (Figge et al., 1988). LT, E1A and c-myc have transforming domains which share amino acid sequence homology and similar secondary structure (Figge et al., 1988). All three proteins complex with the tumor suppressor, retinoblastoma gene product (Rb) (Whyte et al., 1988, DeCaprio et al., 1988, Rustgi et al., 1991), and the Rb binding domains of LT and E1A coincide with their transforming domains. Based on this similarity, it has been thought that LT and E1A transform cells by binding cellular Rb and abrogating its tumor suppressor function. LT, E1A and c-myc are also grouped as immortalization oncogenes as determined by the oncogene cooperation assay using rat embryo fibroblasts (Weinberg, 1985).
In spite of the similarity between the Rb binding domains of LT and E1A, the two proteins differ substantially in other regards. In fact, there is apparently only a short equivalent stretch of acidic amino acids (Figge et al., 1988). This stretch lies between amino acids 106-114 in LT and amino acids 121-139 in E1A. The large T antigen is encoded by the simian virus 40, a member of the polyoma virus family. In contrast, E1A is encoded by adenovirus 5 virus, which is a member of the adenovirus family. LT is 708 amino acids long, while E1A is substantially shorter at 289 or 243 amino acids (for 13S and 12S respectively). LT has been observed to bind directly to certain DNA sequences; however, direct binding of E1A to DNA has not been observed and E1A may instead interact indirectly via a co-activator such as p300 (as discussed in Chen & Hung, 1997). LT binds with the tumor suppressors Rb and also with p53. E1A complexes with Rb but apparently not with p53. E1A has been shown to induce apoptosis in cells, but this has not been demonstrated for LT. Further, LT is an apparent anomaly in the scheme of oncogenic classification. Oncogenes are typically classified as being cytoplasmic or nuclear oncogenes. However, LT, through the actions of a single protein, is able to introduce “nuclear” characteristics such as immortalization as well as “cytoplasmic” characteristics such as anchorage independence in cells (Weinberg, 1985). LT antigen can be found in both the nucleus and at the plasma membrane, and mutations that inhibit the transport of LT into the nucleus appear to reduce its immortalizing ability while leaving intact its effect on anchorage independence and its ability to transform already immortalized cells. Consequently, this oncogene is considered to be a member of both the nuclear and cytoplasmic oncogenic classes, since it its gene product apparently affects these two distinct cellular sites (Weinberg, 1985); which again is unlike E1A.
Despite advances in identifying certain components which contribute to the development of malignancies, it is clear that the art still lacks effective means of suppressing carcinogenesis. Recently, however, M. C. Hung and collaborators have made great advances in the suppression of oncogenic transformation. Some of these advances are described in U.S. Pat. Nos. 5,651,964, 5,641,484, and 5,643,567, the entire text of each being specifically incorporated by reference herein and briefly described below.
Suppression of Oncogenesis
Work by Hung and collaborators has established that the E1A gene can in fact suppress transformation, tumorigenicity and metastasis in a variety of cancers (see, e.g., Yu et al. 1991, 1992 and 1993; and the reviews by Hung et al., 1995, Yu and Hung, 1995, and Mymryk, 1996).
Without wishing to be bound by theory, it appears that there may be more than one pathway or means by which E1A can act to suppress oncogenic transformation. In particular, while Hung and collaborators initially established that E1A can suppress tumor formation in vitro and in vivo in cancers that appear to be associated with an over-expression of an oncogene variously referred to as c-erbB-2, HER-2 or neu (hereinafter the neu oncogene); it appears that E1A can also suppress the oncogenic phenotype in various other cancer cells that do not appear to be associated with an overexpression of neu. Indeed, Frisch et al. have reported that the tumor-suppressing effects of the E1A gene can also be used to convert three unrelated types of human cancer cells (which do not appear to be over-expressing neu) into a non-transform
Chen Hua
Hung Mien-Chie
LaFoc Daniel Nathan Andrew
Loomis Aaron Garth
Paul Ralph Wilfred
Board of Regents , The University of Texas System
Crouch Deborah
Fulbright & Jaworski L.L.P.
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