Ramaswamy Narayanan

Evidence for differential functions of the p50 and p65 subunits of NF-kappa B with a cell adhesion model.

The p50 and p65 subunits of NF-kappa B represent two members of a gene family that shares considerable homology to the rel oncogene. Proteins encoded by these genes form homo- and heterodimers which recognize a common DNA sequence motif. Recent data have suggested that homodimers of individual subunits of NF-kappa B can selectively activate gene expression in vitro. To explore this possibility in a more physiological manner, murine embryonic stem (ES) cells were treated with phosphorothio antisense oligonucleotides to either p50 or p65. Within 5 h after exposure to phosphorothio antisense p65 oligonucleotides, cells exhibited dramatic alterations in adhesion properties. Similar findings were obtained in a stable cell line that expressed a dexamethasone-inducible antisense mRNA to p65. Although antisense oligonucleotides raised against both p50 and p65 elicited a significant reduction in their respective mRNAs, only the cells treated with antisense p50 maintained a normal morphology. However, 6 days following removal of leukemia-inhibiting factor, a growth factor which suppresses embryonic stem cell differentiation, adhesion properties of cells treated with the antisense p50 oligonucleotides were markedly affected. The ability of the individual antisense oligonucleotides to elicit differential effects on cell adhesion, a property dependent upon the stage of differentiation, suggests that the p50 and p65 subunits of NF-kappa B regulate gene expression either as homodimers or as heterodimers with other rel family members. Furthermore, the finding that reduction in p65 expression alone had profound effects on cell adhesion properties indicates that p65 plays an important role in nonstimulated cells and cannot exist solely complexed with the cytosolic inhibitory protein I kappa B.

Identification of a Naturally Occurring Transforming Variant of the p65 Subunit of NF-KB

Transcription factor NF-KB comprises two proteins, p50 and p65, that have sequence similarity to the v-rel oncogene. In primary hematopoietic cell populations an alternatively spliced form of NF-KB p65 mRNA was observed that encoded a protein designated p65Δ. Expression of the p65Δ cDNA in Rat-1 fibroblasts resulted in focus formation, anchorage- independent growth in soft agar, and tumor formation in athymic nude mice, effects not obtained with expression of p65 or a p65Δ mutant that contains a disruption within the transcriptional activation domain. Thus, p65Δ, which associated weakly and interfered with DNA binding by p65, may sequester an essential limiting regulatory factor or factors required for NF-KB function.

Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.

Coordinated expression of intermediate biomarkers for tumorigenic transformation in RAS-transfected mouse mammary epithelial cells

SummaryDeregulated expression of the RAS oncogene is associated with tumorigenic transformation of mammary cells. Because of the complex, multiphasic nature of cancer progression, it is important to systematically identify the biomarkers specific for initiation, promotion, and progression of breast cancer. Mouse mammary epithelial cells (MMEC) were transfected with normal c-Ha-RAS proto oncogene (pH06N) and with mutant c-Ha-RAS oncogene (pH06T). The parental MMEC and the cloned transfectants pH06N1, pH06N2, pH06T1, and pH06T12 were evaluated for the acquisition of transformed characteristics by determining altered cellular metabolism of estradiol, increased ability for anchorage-independent growth, and ability to form tumors at the transplant site in athymic ‘nude’ mice. Persistent, functional integration of c-Ha-RAS was evidenced by the presence of a 1.2 kb c-Ha-RAS transcript in the four transfectants but not in MMEC. All the transfectants also exhibited a substantial increase in the binding of c-Ha-RAS p21 to [α-32P] GTP relative to MMEC (P<0.003). The relative extent of estradiol metabolism leading to the formation of 16α-hydroxyestrone was increased (P<0.004) in all the four transfectants. These four transfectants also showed a 100–400 fold increase in colony forming efficiency in 0.33% agar, relative to MMEC (P<0.0009), and formed rapidly growing tumors within 3–5 weeks of transplantation. Our results demonstrate that i) persistent expression of normal and mutant c-Ha-RAS can bring about tumorigenic transformation of mouse mammary epithelial cells; and ii) alteration in estradiol metabolism and acquisition of anchorage-independent growth precede the emergence of a tumorigenic phenotype. These endpoints therefore may constitute useful intermediate biomarkers to examine oncogene-induced tumorigenic transformation of mammary epithelial cells.

Enhancement by antisense oligonucleotides to NF-kB of the differentiation of HL-60 promyelocytic leukemia cells induced by vitamin D3

We have demonstrated previously that a phosphorothioate antisense oligonucleotide to the p65 subunit of the inducible transcription factor NF-kappaB produced rapid changes in the expression of leukocyte integrin CD11b (Mo 1) and in the adhesion of dimethylsulfoxide (DMSO)-differentiated HL-60 cells stimulated by 12-O-tetradecanoylphorbol 13-acetate. We have also shown that a variety of agents which inhibit NF-kappaB, including vitamin E and related antioxidants, curcumin and several non-steroidal anti-inflammatory agents, significantly enhanced the differentiation of HL-60 leukemia cells when combined with low levels of 1,25-dihydroxyvitamin D3 (vitamin D3). To provide further evidence that interference with the activation of NF-kappaB affects the maturation of HL-60 leukemia cells by creating an environment conducive to terminal differentiation, we measured the effects of phosphorothioate antisense oligonucleotides to the various subunits of NF-kappaB on the differentiation of HL-60 cells produced by low levels of vitamin D3. When used alone these oligonucleotides had no significant effect on the differentiation of HL-60 cells. However, the antisense oligomer to the Rel A subunit of NF-kappaB markedly increased the extent of differentiation produced by low levels of vitamin D3. An enhancement of the differentiation of HL-60 cells induced by vitamin D3 was also obtained by several transcription factor decoys designed to mimic the consensus sequences of genes activated by Rel A. The findings provide additional support for the concept that inhibition of the activation of NF-kappaB may be involved in regulating the entry of promyelocytic leukemia cells into a differentiation pathway.

Antisense oligodeoxynucleotides: Internalization, compartmentalization and non-sequence specificity

Antisense oligodeoxynucleotides are sequence-specific inhibitors of gene expression in both in vitro and in vivo systems. However, their efficacy may be markedly impacted upon by critical aspects of cellular pharmacology. For example, oligodeoxynucleotides must be transported into the cytoplasm and/or nucleus from the extracellular bulk phase. However, the mechanism by which this occurs is not clear. In addition, oligodeoxynucleotides, and particularly phosphorothioates, may interact non-sequence-specifically with cellular components. These issues are discussed with reference to recent findings concerning antisense inhibition of the NF-κB nuclear transcriptional regulatory factor.

Evaluation of the role of extracellular matrix proteins, polyunsaturated fatty acids and C-MYC expression in the inhibition of the serum-free growth of epithelial cells by TGF-β1

SummaryNovel or modified serum-free media were developed for the anchorage-dependent growth of nontransformed murine mammary epithelial cells (MMEC) and Balb/MK murine keratinocytes respectively. Growth rates for both cell lines were similar in serum-containing and serum-free media. The serum-free media were used to evaluate potential mechanisms of epithelial cell growth regulation by type 1 transforming growth factor β(TGF-β1). The growth of MMEC and Balb/ MK cells was reversibly inhibited 40–65% in a time- and dose-dependent fashion by TGF-β1 under both serum-containing and serum-free conditions. Constitutive over-expression of a stranfected c-myc oncogene inMMEC did not result in loss of sensitivity to growth inhibition by TGF-β1. In addition, Balb/MK and MMEC growth inhibition by TGF-β1 was not potentiated by polynsaturated fatty acids or reversed by vitamin E. Expgenous type V collagen was able to mimic the inhibitory effects of TGF-β1 on the serum-free growth of Balb/MK and MMEC. In contrast, collagen type I and IV, fibronectin and laminin did not inhibit the growth of these cells. The type V collagen used was not contaminated with TGF-β, and subsaturating, but not saturating concentrations of type V collagen and TGF-β1 were additive with respect to Balb/MK and MMEC growth inhibition. These results demonstrate that nontransformed epithelial cell growth inhibition by TGF-β1 is mediated by mechanisms distinct from those observed with certain carcinoma and melanoma cells. Our results also suggest the possible involvement of type V collagen in Balb/MK and MMEC growth inhibition by TGF-β1.

Antisense RNA to the Putative Tumor Suppressor Gene “Deleted in Colorectal Cancer” Transforms Fibroblasts

Allelic deletions involving chromosome 18q occur in over 70% of colorectal cancers.’ A candidate suppressor gene, termed “deleted in colorectal cancer” (DCC), has been identified in this region.2 DCC expression was detected in many normal tissues (e.g., brain and colonic mucosa), but it was absent or diminished in most colorectal carcinomas.* The predicted amino acid sequence of the DCC cDNA specified a protein with sequence homology to neural cell adhesion molecules and various other related cell surface glycoproteins.* We attempted to establish the tumor suppressor function of DCC by inhibiting its expression in normal cells using antisense RNA technology. We chose a dexamethasone-inducible vector, pMAM Neo CAT (MNC), to produce an inducible chimeric antisense RNA to DCC.3 When Rat-1 cells were transfected with the activated ras, MNC (vector), DCC,S and DCCAS constructs, stable G-4MR clones were readily established for the ras, MNC, or DCCS transfectants but not for the DCCAS transfectant. We reasoned that expression of antisense RNA to DCC might interfere with the cells’ ability to attach to the substratum. Subsequently, we established several independent G-418R clones from DCCAS-transfected cells grown in dishes coated with an extracellular matrix component, laminin. The DCCAS transfectants showed 2.5to 3-fold increased saturation density, faster growth rate, and anchorage independence in a dexamethasone-dependent manner as well as in vivo tumorigenicity in nude mice. These DCCAS transfectants showed a pronounced reduction in DCC gene expression in a dexamethasone-dependent manner as monitored by quantitative reverse transcriptase PCR.3

Antisense rel A in Cancer.

NFKB: is a pleiotropic transcription factor that participates in the induction of various cellular and viral genes (for a review, see refs. 1 and 2). The principal form of this active complex is composed of two polypeptides, p65 (recently renamed rel A) and p50 (recently renamed NFKB1) (3, 4). Both of these sub-units belong to the rel famtly of transcription factors with homology in the amino terminus (5, 6). Since the original identification of these factors, several others have been identified in this family of transcription factors: P49, c-rel, p100, rel B, dorsal, Bcl-3, and p105 (1). The NF-kheterodimer is associated in the cytoplasm with the IkB subunit, which sequesters this factor in an inactive state (7). On activation by various stimuli, the IkB subunit separates from the active NFheterodimer, and the active complex is translocated to the nucleus where it binds to its nuclear DNA target sequence (Fig. 1). The principal cause for disassociation of IkB from NFis phosphorylation (8), but it may involve proteolysis of IkB (9) Several autoregulatory loops have been proposed for the involvement of NF-kB in the regulation of the inhibitor IkB. Furthermore, NF-kB has been shown to regulate the transcription of IkB and NFKB1 (10-12). Fig. 1. NF-K: B transcription factor. This figure depicts the basis for the function of the NF-K: B transcription factor. The principal form of the NF-K: B transcription complex is as a heterodimer composed of the two subunits: rel A and NF-K: B 1. The inactive complex is sequestered in the cytoplasm associated with the inhibitor protein, IK: B. On activation, the IK: B protein separates from the active complex through a mechanism involving the phosphorylation and proteolysis Of IK: B. The active NF-K: B complex is then translocated to the nucleus where it binds DNA in a sequence-specific manner and interacts with the transcriptional machinery to modulate the rate of transcription of target genes.