Reexpression of the retinoblastoma protein in tumor cells induces senescence and telomerase inhibition.

Normal human diploid cells senesce in vitro and in vivo after a limited number of cell divisions. This process known as cellular senescence is an underlying cause of aging and a critical barrier for development of human cancers. We demonstrate here that reexpression of functional pRB alone in RB/p53-defective tumor cells via a modified tetracycline-regulated gene expression system resulted in a stable growth arrest at the G0/G1 phase of the cell cycle, preventing tumor cells from entering S phase in response to a variety of mitogenic stimuli. These cells displayed multiple morphological changes consistent with cellular senescence and expressed a senescence-associated beta-galactosidase biomarker. Further studies indicated that telomerase activity, which was assumably essential for an extended proliferative life-span of neoplastic cells, was abrogated or repressed in the tumor cell lines after induction of pRB (but not p53) expression. Strikingly, when returned to an non-permissive medium for pRB expression, the pRB-induced senescent tumor cells resumed DNA synthesis, attempted to divide but most died in the process, a phenomenon similar to postsenescent crisis of SV40 T-antigen-transformed human diploid fibroblasts in late passage. These observations provide direct evidence that overexpression of pRB alone in RB/p53-defective tumor cells is sufficient to reverse their immortality and cause a phenotype that is, by all generally accepted criteria, indistinguishable from replicative senescence. The results suggest that pRB may play a causal role in the intrinsic cellular senescence program.

Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro.

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, the capacity of tumor cells to migrate or invade was quantitated by the Boyden chamber assay. The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the lung, breast and bladder. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RB revertants than from the RB-reconstituted RB+ cell lines penetrated through the Matrigel (P<0.001, two-tailed t-test), although both RB+ and RB- cells migrated at approximately the same rate on uncoated polycarbonate filters in the Boyden chambers. Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 re-expression effectively suppressed tumor formation in nude mice of the RB-/p53null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene. These studies provided an insight into the broader biological basis of the RB-mediated tumor suppression in RB-defective tumor cells.

Enhanced tumor suppressor gene therapy via replication-deficient adenovirus vectors expressing an N-terminal truncated retinoblastoma protein.

The preclinical studies presented here demonstrate that treatment of human non-small cell lung carcinoma and bladder carcinoma cells by a recombinant adenovirus vector, AdCMVpRB94, expressing the N-terminal truncated retinoblastoma (RB) protein (pRB94) completely suppressed the tumorigenicity of the treated tumor cells in nude mice. Furthermore, gene therapy of established human RB- and RB+ bladder xenograft cancers in nude mice by AdCMVpRB94 resulted in regression of the treated tumors. Of note, although both the full-length and the truncated forms of the RB protein, when overexpressed in tumor cells via replication-deficient adenovirus vectors, were capable of suppression of tumor growth, the pRB94 was evidently more potent than the full-length RB protein.

Expression of N-acetylglucosaminyltransferase V mRNA in mammalian tissues and cell lines.

The expression of N-acetylglucosaminyltransferase V (Glc-NAc-T V) is increased in many oncogenically transformed cells and in cell lines which are induced to proliferate. In order to characterize the regulation of GlcNAc-T V at the molecular level, we have examined the expression of Glc-NAc-T V mRNAs in a number of mouse tissues and in several human and rodent cell lines. The GlcNAc-T V mRNA is expressed in different amounts in the various mouse tissues, with the greatest amount observed in brain, followed by thymus, kidney, lung, intestine, heart and stomach, and no transcripts detected in liver or skeletal muscle. Measurements of GlcNAc-T V enzymatic activity, by contrast, show brain to have lower levels of activity than several of the other tissues, suggesting possible post-translational regulation. We find that there are two GlcNAc-T V transcripts in most of the RNAs analysed. The rodent cell lines all express both a 7.5 and a 9.5 kb mRNA, with the smaller transcript being more abundant. The human cells have mRNAs of 4.5 and 9.5 kb. Both mRNAs are expressed in HepG2 and MCF-7 cells, while A431 cells express only the 4.5 kb mRNA and HL60 cells express only the 9.5 kb transcript. Furthermore, only the 9.5 kb mRNA appears to be increased, along with activity, when HepG2 cells are stimulated to proliferate, suggesting that the two mRNAs may be under different regulatory controls. Also, a GlcNAc-T V-deficient, mutant lymphoma cell line, PHAR2.1, was found to express mRNAs which are larger than the normal mRNAs, possible due to an insertion or aberrant splicing, resulting in a defective mRNA.

Isolation, characterization, and expression of a cDNA encoding N-acetylglucosaminyltransferase V.

A cDNA clone for the complete coding sequence for alpha-1,3(6)-mannosylglycoprotein beta-1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155) was isolated and expressed in COS-7 cells. Degenerate oligonucleotide primers for polymerase chain reaction were synthesized based on the amino acid sequence of three tryptic peptides isolated from affinity-purified GlcNAc-T V. Polymerase chain reaction amplimers were isolated from rat and mouse mRNA. A cDNA-encoding full-length enzyme was isolated from a rat 1 cell (EJ-ras-transformed) library and sequenced. Transient expression of this clone in COS-7 cells, followed by enzymatic activity assays, demonstrated that this cDNA sequence encodes GlcNAc-T V. Northern analysis of rat kidney mRNA revealed a single band corresponding to a length of about 7 kilobases. Sequence analysis of the cDNA clone demonstrated an open reading frame that encoded a type II membrane protein of 740 amino acids.