Pdcd4 repression of lysyl oxidase inhibits hypoxia-induced breast cancer cell invasion.

Metastasis to bone, liver and lungs is the primary cause of death in breast cancer patients. Our studies have revealed that the novel tumor suppressor Pdcd4 inhibits breast cancer cell migration and invasion in vitro. Loss of Pdcd4 in human nonmetastatic breast cancer cells increased the expression of lysyl oxidase (LOX) mRNA.

Translation inhibitor Pdcd4 is targeted for degradation during tumor promotion.

Inactivation of tumor suppressors is among the rate-limiting steps in carcinogenesis that occur during the tumor promotion stage. The translation inhibitor programmed cell death 4 (Pdcd4) suppresses tumorigenesis and invasion. Although Pdcd4 is not mutationally inactivated in human cancer, the mechanisms controlling Pdcd4 inactivation during tumorigenesis remain elusive.

A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A.

An alpha-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding.

Transformation nonresponsive cells owe their resistance to lack of p65/nuclear factor-kappaB activation.

Clonal variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to tumor promoter-induced neoplastic transformation exhibit differential activator protein-1 (AP-1) response. Transactivation of AP-1 appears to be necessary but not sufficient to promote transformation in JB6 cells. Inhibition of AP-1 is invariably accompanied by inhibition of nuclear factor-kappaB (NF-kappaB) when transformation is suppressed, suggesting that NF-kappaB may also play a role in neoplastic transformation.

cDNA cloning and mapping of mouse pleckstrin (Plek), a gene upregulated in transformation-resistant cells.

Changes that occur during tumor promotion, the rate-limiting phase of multistep carcinogenesis, may offer the best targets for prevention of cancer or reversal of early disease. The murine epidermal JB6 promotion-sensitive (P+) and -resistant (P-) cell lines provide a cell culture model for tumor promoter-induced neoplastic transformation ideally suited to the identification of molecular events that mediate or inhibit transformation. A differential display comparison of P+ and P- cell mRNAs yielded seven differentially expressed sequences.

Differentially expressed protein Pdcd4 inhibits tumor promoter-induced neoplastic transformation.

An mRNA differential display comparison of mouse JB6 promotion-sensitive (P+) and -resistant (P-) cells identified a novel gene product that inhibits neoplastic transformation. The JB6 P+ and P- cells are genetic variants that differ in their transformation response to tumor promoters; P+ cells form anchorage-independent colonies that are tumorigenic, and P- cells do not. A differentially displayed fragment, A7-1, was preferentially expressed in P- cells at levels >/=10-fold those in P+ cells, making its mRNA a candidate inhibitor of neoplastic transformation.

Specific methylation events contribute to the transcriptional repression of the mouse tissue inhibitor of metalloproteinases-3 gene in neoplastic cells.

The tissue inhibitor of metalloproteinases-3 (TIMP-3) gene is specifically down-regulated in neoplastic cells of the mouse JB6 progression model, suggesting a role for TIMP-3 inactivation in neoplastic progression. On the basis of 5-azacytidine reversal, the mechanism for this down-regulation appears to involve changes in the methylation state of the TIMP-3 promoter. Although total genomic methylation levels are comparable, specific differences in the methylation of the TIMP-3 promoter were observed between preneoplastic and neoplastic JB6 cells at three Hpall sites, with preneoplastic cells being less methylated.

Molecular cloning of mouse tissue inhibitor of metalloproteinases-3 and its promoter. Specific lack of expression in neoplastic JB6 cells may reflect altered gene methylation.

Mouse tissue inhibitor of metalloproteinases-3 (mTIMP-3), a gene specifically not expressed in neoplastic JB6 cells, have been isolated recently through the use of the mRNA differential display technique (Sun, Y., Hegamyer, G., and Colburn, N.

Status of the mdm-2 and waf-1 genes in mouse epidermal jb6 variants harboring wild-type p53 - a p53-independent induction of waf-1.

Mutational inactivation of the p53 tumor suppressor gene has been found not to be involved in preneoplastic-to-neoplastic progression in mouse JB6 variants. To examine the role of an inactivated p53 pathway in this tumor promotion/progression model, we have studied the possible alteration of the MDM-2 oncogene, a gene whose product binds to and inactivates p53, and WAF-1 tumor suppressor gene, a gene transcriptionally controlled by p53 that mediates p53 tumor suppression. Alteration of either of these two genes might mimic p53 inactivation in cells expressing wild-type p53.

Molecular cloning of five messenger RNAs differentially expressed in preneoplastic or neoplastic JB6 mouse epidermal cells: one is homologous to human tissue inhibitor of metalloproteinases-3.

To better understand the molecular mechanism of multistage carcinogenesis, we have attempted to identify putative oncogenes and/or tumor suppressor genes involved in preneoplastic-to-neoplastic progression of mouse epidermal JB6 variants. The JB6 variants consist of P- (promotion resistant), P+ (promotion sensitive), and Tx [transformed; both apoptosis-sensitive (A(s)) and apoptosis-resistant (Ar)] cells, representing progression from early to late stages of carcinogenesis. By using the newly developed differential mRNA display technique, we have isolated five clones from these JB6 variants.

Alterations of the p53 tumor-suppressor gene in transformed mouse liver cells.

Mutational inactivation of p53, a potential tumor-suppressor gene, has been found in many tumors of humans as well as rodents. The p53 status in normal and transformed mouse liver cell lines has, however, not been investigated. We examined possible point mutations and compared mRNA and protein expression of the p53 gene in normal vs.

Sequence and localization of a novel FK506-binding protein to mouse chromosome 11.

We have isolated a unique gene from a mouse JB6 epidermal cell cDNA expression library, termed FKBPRP, that codes for a protein having domains that share between 37 and 44% amino acid sequence identity and 60% similarity with members of the family of FK506-binding proteins. The FKBPRP protein has three repeats contained within its sequence that share between 48 and 58% identity to each other. We have localized the FKBPRP gene to mouse Chromosome 11, and crosses of different murine strains provided the gene order centromere--FKBPRP-Int-4-Pkca-Es-3.

Sequence of manganese superoxide dismutase-encoding cDNAs from multiple mouse organs.

Manganese superoxide dismutase (MnSOD)-encoding cDNAs from multiple mouse organs, including liver, kidney, brain, spleen and heart, show three sequence differences compared with the previously published mouse placenta MnSOD cDNA. The differences cause substitutions of Val(-7)-->Gly (GTG-->GGT) and Met(114)-->Val (ATG-->GTG) and a G746 deletion in the 3' untranslated region. The analysis, by reverse transcriptase-polymerase chain reaction-direct sequencing, of BALB/c mouse placenta RNA revealed the same sequence in the placenta MnSOD as that from multiple mouse organs.

Nasopharyngeal carcinoma shows no detectable retinoblastoma susceptibility gene alterations.

Since multistage carcinogenesis is frequently associated with the loss of suppressor gene activity, and since in over 90% of cases of nasopharyngeal carcinoma (NPC) p53 alterations are not involved [Sun, Y., Hegamyer, G.H.

No point mutation of Ha-ras or p53 genes expressed in preneoplastic-to-neoplastic progression as modeled in mouse JB6 cell variants.

Alterations of the p53 gene have been found in many human and animal tumors, and ras mutations have been seen somewhat less frequently. Loss of p53 tumor suppressor activity has been implicated in multistage tumor promotion/progression after initiation by Ha-ras or Ki-ras activation. Whether p53 or ras alterations occur during promotion of neoplastic transformation in JB6 cells has not been reported.

An infrequent point mutation of the p53 gene in human nasopharyngeal carcinoma.

Point mutations in the p53 gene have been detected in a variety of human cancers; the mutations are clustered in four "hot-spots" located in the coding region of exons 5, 7, and 8, which coincide with the four most highly conserved regions of the gene. We report the finding of a heterozygous G----C mutation at codon 280 (exon 8), position 2, of the p53 gene in a nasopharyngeal carcinoma (NPC) cell line, originating from Guangdong, a province in the People's Republic of China that leads the world in NPC incidence. A survey of nasopharyngeal tissues and NPC biopsies revealed that 1 out of 12 NPC samples from Hunan, another province in the People's Republic of China with high NPC incidence, had the same heterozygous mutation at codon 280 of p53, and none of 10 biopsies from Taiwan showed a mutation within exons 5-8 of the p53 gene.

A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.

Isolation and partial characterization of a transformation-associated sequence from human nasopharyngeal carcinoma.

A transforming activity associated with Chinese nasopharyngeal carcinoma (NPC) cell line CNE2 DNA has been identified by transfer into nontransformed promotion-sensitive mouse JB6(P+) C141 cells. To clone this transformation-associated sequence, we carried out three cycles of transfection, followed by cloning of anchorage-independent transformants in soft agar. A tertiary CNE/JB6 clonal transfectant cell line 625 whose DNA showed transforming activity, as indicated in both soft-agar assay and nude-mice implantation, was used to make a genomic library in the vector lambda dash.

Deletion mapping of tumor promotion-susceptibility gene pro1 implicates an RNA polymerase III transcription unit.

The murine gene pro1 has been cloned from JB6 epidermal cell lines that are sensitive to neoplastic transformation by tumor promoters. Insensitive JB6 variants acquire susceptibility to neoplastic transformation by tumor promoters when transfected with pro1. The repetitive nature of pro1 was indicated by sequence and Southern analysis.

Cloning and characterization of putative genes that specify sensitivity to neoplastic transformation by tumor promoters.

Two putative genes (termed pro 1 and pro 2) specifying sensitivity to induction of neoplastic transformation by TPA in mouse epidermal JB6 cells were cloned by sib selection from a size-selected genomic library of clonal cells sensitive to promotion of transformation. By restriction analysis, heteroduplex analysis, direct hybridization, and sequencing, the putative genes are different from and have no homology to known oncogenes. Both genes are independently and equally active as total DNA in the transfection assay.

A transforming activity not detected by DNA transfer to NIH 3T3 cells is detected by JB6 mouse epidermal cells.

Transfection of four different mouse epidermal tumor cell DNAs into NIH 3T3 cells yielded neither morphologically altered foci nor anchorage independence. However, promotion-sensitive, but not promotion-insensitive, JB6 mouse epidermal cell lines were permissive for the expression of anchorage independence after transfection of DNA from three of these tumor cell lines. This transforming activity and the promotion-sensitive activity that confers sensitivity to promotion of transformation show differences in restriction enzyme sensitivity.

Role of specific membrane and genetic changes in the mechanism of tumour promotion. Studies with promoter-resistant variants.

Several cell culture model systems in current use for studying tumour promotion mechanism are reviewed briefly. The conclusions that can be drawn from studies with the JB6 mouse epidermal system are summarized. Promoter-induced mitogenic stimulation, epidermal growth factor receptor binding and stimulated hexose transport are apparently not required for promotion of neoplastic transformation in JB6 cells by phorbol esters and other promoters.

Phorbol diester and epidermal growth factor receptors in 12-O-tetradecanoylphorbol-13-acetate-resistant and -sensitive mouse epidermal cells.

Several cell variants have been isolated from promotable mouse JB6 epidermal cells which are resistant either to mitogenic stimulation at quiescence or to promotion of anchorage independence by 12-O-tetradecanoylphorbol-13-acetate (TPA). Such resistant variants would be expected to lack one or more steps in the TPA response pathway leading to mitogenesis or promotion of tumor cell phenotype. This report is concerned with determining whether resistance is attributable to lack of receptors for phorbol diesters or epidermal growth factor (EGF, a potential mediator) or to absence of receptor down modulation following ligand binding.