Evaluation of horn bud wound healing following cautery disbudding of preweaned dairy calves treated with aluminum-based aerosol bandage.

Pain management during and following disbudding procedures has been studied extensively, though few studies have evaluated wound healing following cautery disbudding in dairy calves. The purpose of this study was to observe wound healing following cautery disbudding with or without treatment using a topical aluminum-based aerosol bandage (ALU) in preweaned dairy calves. Dairy calves were disbudded within the first 3 wk of life using a standard cautery disbudding protocol. The ALU treatment was randomly allocated to the right or left horn bud within each animal. The outcomes measured were lesion score (LS) and wound diameter (WD). The LS was evaluated on a scale of 1 to 3, with LS = 1 representing normal healing without a scab or exudate, LS = 2 having the presence of a scab, and LS = 3 showing the presence of wound exudate. Lesion score and WD were evaluated on a weekly basis following dehorning for 3 wk. A total of 209 animals completed the study. No difference was observed in LS between groups during the first 2 wk postdisbudding, but the proportion of LS = 3 on wk 3 postdisbudding was greater for the control group when compared with ALU (17 vs. 8%, respectively). During wk 1 and 2 postdisbudding, the odds of having delayed healing, or a LS ≥2, were similar for both groups. However, the odds tended to be different at wk 3 postdisbudding with control disbudding sites being 1.42 times more likely to have delayed healing than ALU. In wk 3, WD was 1 mm smaller in the treatment group compared with the control, and treatment decreased diameter over time compared with controls. Overall, once abnormal wound healing was observed, the likelihood of having abnormal wound healing the following week was increased. However, treatment with ALU diminished this effect on delayed healing during the follow-up period. Based on these results, the use of ALU improved wound healing following cautery disbudding of preweaned dairy calves.

DNA demethylation and tri-methylation of H3K4 at the TACSTD2 promoter are complementary players for TROP2 regulation in colorectal cancer cells.

TROP2 is a powerful cancer driver in colorectal cancer cells. Divergent epigenetic regulation mechanisms for the corresponding TACSTD2 gene exist such as miRNAs or DNA methylation. However, the role of TACSTD2 promoter hypermethylation in colorectal cancer has not been investigated yet. In this study, TROP2 expression strongly correlated with promoter methylation in different colorectal tumor cell lines. Treatment with 5-Azacytidine, a DNMT1 inhibitor, led to demethylation of the TACSTD2 promoter accompanied by an increase in TROP2 protein expression. TROP2 expression correlated with promoter methylation in vivo in human colon tumor tissue, thereby verifying promoter methylation as an important factor in the regulation of TROP2 expression in colorectal cancer. When performing a ChIP-Seq analysis in HCT116 and HT29 cells, we found that TACSTD2 promoter demethylation was accompanied by tri-methylation of H3K4. In silico analysis of GSE156613 data set confirmed that a higher binding of histone mark H3K4me3 around the TACSTD2 promoter was found in TACSTD2 high expressing tumors of colon cancer patients compared to the corresponding adjacent tumor tissue. Moreover, the link between TROP2 and the H3K4me3 code was even evident in tumors showing high intratumoral heterogeneity for TROP2 staining. Our data provide novel evidence for promoter demethylation and simultaneous gains of the active histone mark H3K4me3 across CpG-rich sequences, both being complementary mechanisms in the transcriptional regulation of TACSTD2 in colon cancer. The functional consequences of TROP2 loss in colorectal cancer needs to be further investigated.

FRA3B and other common fragile sites: the weakest links.

In 1979, the first chromosome alteration associated with familial cancer was reported. Five years later, a fragile site was observed in the same chromosome region. The product of the fragile histidine triad (FHIT) gene, which encompasses this fragile site, is partially or entirely lost in most human cancers, indicating that it has a tumour-suppressor function. Inactivation of only one FHIT allele compromises this suppressor function, indicating that a 'one-hit' mechanism of tumorigenesis is operative. Are genes disrupted at other fragile sites? And, are these genes also tumour suppressors?

A murine teratocarcinoma stem cell line carries suppressed oncogenic virus genomes.

Murine teratocarcinoma stem cells are nonpermissive for productive infection by a variety of DNA (polyoma and SV40 virus) and RNA (murine leukemia and sarcoma virus) tumor viruses whereas differentiated murine cells derived from the stem cells are permissive for productive (or abortive in the case of SV40) infection by these same viruses. The block to productive infection by these oncogenic viruses is at a postpenetration step in the replication cycle of these viruses but the precise level of the block has not been established for any of these viruses. In this report we describe teratocarcinoma-derived stem and differentiated cell lines which should be especially useful in determining the level of the block to replication of ecotropic murine leukemia virus in murine teratocarcinoma stem cells. The stem cell line, OTT6050AF1 BrdU, which is completely nonpermissive to productive infection by Moloney murine leukemia virus and consists of 97% pluripotent stem cells, contains DNA copies of an RNA tumor virus which is indistinguishable from the N-tropic murine leukemia virus of AKR mice. The stem cells are negative for expression of viral reverse transcriptase, p30 and gp69/71 and no virus is found by XC plaque assay or other biological tests. Differentiated cells established from the same teratocarcinoma tumor are 100% positive for viral gp69/71, p30, and produce large amounts of reverse transcriptase activity and N-tropic virus as detected by biological assay. The virus isolated from the differentiated cells is closely related, if not identical to AKR N-tropic virus by nucleic acid hybridization studies and is thus not an endogenous virus of the 129 strain of mice. The teratocarcinoma tumor from which the cell lines were established had been carried in 129 mice and perhaps at some time in the mouse passage history the tumors were infected (nonproductively) with the N-tropic virus. Regardless of the origin of this viral DNA, the OTT6050A derived stem and differentiated cell lines should be extremely useful in defining in stem cells the step at which ecotropic murine leukemia virus replication is blocked.

Correction: Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential.

After publication of this Article the authors noticed errors in several figures. In Fig. 2b the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 1b, therefore the Gapdh panels should be the same in both figures. In Fig. 3b the Gapdh panels for Ad-Fhit-wt and Ad-Fhit-Y114F are incorrect and have been replaced with scans from original films. In Fig. 4A the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 3b, therefore the Gapdh panels should be the same in both figures. In Fig. 4Bb the Gapdh panels for Fhit siRNA were incorrect and have been replaced with scans from original films. All resupplied figures are provided below. In Fig. 5C several panels are incorrect. The Authors were unable to locate the original films for all of these panels so Fig. 5c has been deleted. The scientific conclusions of this paper have not been affected.

Coexpression of translocated and normal c-myc oncogenes in hybrids between Daudi and lymphoblastoid cells.

Mechanisms that affect the transcription of the c-myc oncogene take part in the development of B-cell neoplasias such as Burkitt's lymphoma. Daudi Burkitt lymphoma cells, which express only the translocated c-myc oncogene, were hybridized with human lymphoblastoid cells, which express the normal c-myc gene; the hybrids were phenotypically lymphoblastoid and expressed both the translocated and the normal c-myc gene. This result contrasts with the findings that the decapitated c-myc gene, translocated to an immunoglobulin switch mu or alpha region, is transcriptionally silent in lymphoblastoid hybrids. Thus, there may be at least two distinct enhancer-like elements capable of deregulating c-myc transcription in lymphomas and leukemias with t(8;14) chromosome translocations. In addition, since the Daudi X lymphoblastoid hybrids express both the translocated and the normal c-myc gene, the c-myc gene product does not autoregulate c-myc transcription.

Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV.

Cells infected with the human immunodeficiency virus (HIV) show decreased expression of the 58-kilodalton T4 (CD4) antigen on their surface. In this study, the effect of HIV infection on the synthesis of T4 messenger RNA (mRNA) and protein products was evaluated in T-cell lines. Metabolically labeled lysates from the T4+ cell line Sup-T1 were immunoprecipitated with monoclonal antibodies to T4. Compared with uninfected cells, HIV-infected Sup-T1 cells showed decreased amounts of T4 that coprecipitated with both the 120-kilodalton viral envelope and the 150-kilodalton envelope precursor molecules. In four of five HIV-producing T-cell lines studied, the steady-state levels of T4 mRNA were also reduced. Thus, the decreased T4 antigen on HIV-infected cells is due to at least three factors: reduced steady-state levels of T4-specific mRNA, reduced amounts of immunoprecipitable T4 antigen, and the complexing of available T4 antigen with viral envelope gene products. The data suggested that the T4 protein produced after infection may be complexed with viral envelope gene products within infected cells. Retroviral envelope-receptor complexes may thus participate in a general mechanism by which receptors for retroviruses are down-modulated and alterations in cellular function develop after infection.

elk, tissue-specific ets-related genes on chromosomes X and 14 near translocation breakpoints.

The myb-ets-containing acute leukemia virus, E26, transforms myeloblasts and erythroblasts in culture and causes a mixed erythroid and myeloid leukemia in chicks. Genes (ets-1, ets-2, and erg) with variable relatedness to the v-ets oncogene of the E26 virus have been identified, cloned, and characterized in several species. Two new members (elk-1 and elk-2) of the ets oncogene superfamily have now been identified. Nucleotide sequence analysis of the elk-1 cDNA clone revealed that this gene encodes a 428-residue protein whose predicted amino acid sequence showed 82% similarity to the 3' region of v-ets. The elk or related sequences appear to be transcriptionally active in testis and lung. The elk cDNA probe detects two loci in the human genome, elk-1 and elk-2, which map to chromosome regions Xp11.2 and 14q32.3, respectively. These loci are near the translocation breakpoint seen in the t(X;18) (p11.2;q11.2), which is characteristic of synovial sarcoma, and the chromosome 14q32 breakpoints seen in ataxia telangiectasia and other T cell malignancies. This suggests the possibility that rearrangements of elk loci may be involved in pathogenesis of certain tumors.

The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000-dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM-CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome.

Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential.

The Fhit tumor suppressor binds and hydrolyses diadenosine polyphosphates and the Fhit-substrate complex has been proposed as a proapoptotic effector, as determined by infection of susceptible cancer cells with adenoviruses carrying wild-type fragile histidine triad (FHIT) or catalytic site mutants. The highly conserved Fhit tyrosine 114 (Y114), within the unstructured loop C-terminal of the catalytic site, can be phosphorylated by Src family tyrosine kinases, although endogenous phospho-Fhit is rarely detected. To explore the importance of Y114 and identify Fhit-mediated signaling events, wild-type and Y114 mutant FHIT-expressing adenoviruses were introduced into two human lung cancer cell lines. Caspase-dependent apoptosis was effectively induced only by wild-type but not Y114 mutant Fhit proteins. By expression profiling of FHIT versus mutant FHIT-infected cells, we found that survivin, an Inhibitor of Apoptosis Protein (IAP) family member, was significantly decreased by wild-type Fhit. In addition, Fhit inhibited activity of Akt, a key effector in the phosphatidylinositol 3-OH kinase (PI3K) pathway; loss of endogenous Fhit expression caused increased Akt activity in vitro and in vivo, and overexpression of constitutively active Akt inhibited Fhit-induced apoptosis. The results indicate that the Fhit Y114 residue plays a critical role in Fhit-induced apoptosis, occurring through inactivation of the PI3K-Akt-survivin signal pathway.

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

Glycoprotein Ib beta (GPIb beta) exists in platelets disulfide-linked to glycoprotein Ib alpha (GPIb alpha), a major receptor for von Willebrand factor. Both GPIb alpha and GPIb beta are expressed in endothelial cells (EC). While the GPIb alpha mRNA and protein appear similar in platelets and EC, EC GPIb beta mRNA is larger than platelet GPIb beta and encodes a larger protein. We have cloned and sequenced EC GPIb beta cDNA and report a 2793-nucleotide sequence which contains a 411-amino acid open reading frame. The EC sequence contains all of the platelet cDNA sequence and all but three amino acids of the primary translation product. Like the genes encoding GPIb alpha, GPIX, and GPV, the GPIb beta gene appears simple in structure. Using human hamster hybrids, we have localized the GPIb beta gene to chromosome 22pter-->22q11.2. When we examined poly (A)+ RNA from several human tissues for GPIb beta mRNA expression, we found that GPIb beta mRNA was expressed in a variety of tissues but was most abundant in heart and brain, while GPIb alpha and GPIX mRNA expression was found only in lung and placenta at very low levels. The broad distribution of GPIb beta mRNA suggests that it may be playing a role different than or additional to its function in platelets.

Influence of a nonfragile FHIT transgene on murine tumor susceptibility.

FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction.

Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice.

Leukemia results from the accumulation of multiple genetic alterations that disrupt the control mechanisms of normal growth and differentiation. The use of inbred mouse strains that develop leukemia has greatly facilitated the identification of genes that contribute to the neoplastic transformation of hematopoietic cells. BXH-2 mice develop myeloid leukemia as a result of the expression of an ecotropic murine leukemia virus that acts as an insertional mutagen to alter the expression of cellular proto-oncogenes. We report the isolation of a new locus, Meis1, that serves as a site of viral integration in 15% of the tumors arising in BXH-2 mice. Meis1 was mapped to a distinct location on proximal mouse chromosome 11, suggesting that it represents a novel locus. Analysis of somatic cell hybrids segregating human chromosomes allowed localization of MEIS1 to human chromosome 2p23-p12, in a region known to contain translocations found in human leukemias. Northern (RNA) blot analysis demonstrated that a Meis1 probe detected a 3.8-kb mRNA present in all BXH-2 tumors, whereas tumors containing integrations at the Meis1 locus expressed an additional truncated transcript. A Meis1 cDNA clone that encoded a novel member of the homeobox gene family was identified. The homeodomain of Meis1 is most closely related to those of the PBX/exd family of homeobox protein-encoding genes, suggesting that Meis1 functions in a similar fashion by cooperative binding to a distinct subset of HOX proteins. Collectively, these results indicate that altered expression of the homeobox gene Meis1 may be one of the events that lead to tumor formation in BXH-2 mice.

Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases.

The tyrosine phosphatase RPTP gamma is a candidate tumor suppressor gene since it is located on human chromosome 3p14.2-p21 in a region frequently deleted in certain types of renal and lung carcinomas. In order to evaluate its oncogenic potential and to explore its normal in vivo functions, we have isolated cDNAs and deduced the complete sequences of both human and murine RPTP gamma. The murine RPTP gamma gene has been localized to chromosome 14 to a region syntenic to the location of the human gene. Northern (RNA) blot analysis reveals the presence of two major transcripts of 5.5 and 8.5 kb in a variety of murine tissues. In situ hybridization analysis reveals that RPTP gamma mRNA is expressed in specific regions of the brain and that the localization of RPTP gamma changes during brain development. RPTP gamma is composed of a putative extracellular domain, a single transmembrane domain, and a cytoplasmic portion with two tandem catalytic tyrosine phosphatase domains. The extracellular domain contains a stretch of 266 amino acids with striking homology to the zinc-containing enzyme carbonic anhydrase (CAH), indicating that RPTP gamma and RPTP beta (HPTP zeta) represent a subfamily of receptor tyrosine phosphatases. We have constructed a model for the CAH-like domain of RPTP gamma based upon the crystal structure of CAH. It appears that 11 of the 19 residues that form the active site of CAH are conserved in RPTP gamma. Yet only one of the three His residues that ligate the zinc atom and are required for catalytic activity is conserved. On the basis of this model we propose that the CAH-like domain of RPTP gamma may have a function other than catalysis of hydration of metabolic CO2.

Retroposition in a family of carcinoma-associated antigen genes.

The gene encoding the carcinoma-associated antigen defined by the monoclonal antibody GA733 is a member of a family of at least two type I membrane proteins. This study describes the mechanism of evolution of the GA733-1 and GA733-2 genes. A full-length cDNA clone for GA733-1 was obtained by screening a human placental library with a genomic DNA probe. Comparative analysis of the cDNA sequence with the previously determined genomic sequence confirmed that GA733-1 is an intronless gene. The GA733-2 gene encoding the monoclonal antibody-defined antigen was molecularly cloned with a cDNA probe and partially sequenced. Comparison of GA733-2 gene sequences with the previously established cDNA sequence revealed that this gene consists of nine exons. The putative promoter regions of the GA733-1 and GA733-2 genes are unrelated. These findings suggest that the GA733-1 gene was formed by the retroposition of the GA733-2 gene via an mRNA intermediate. Prior to retroposition, the GA733-2 gene had been affected by exon shuffling. Analysis of GA733-2 exons revealed that many delineate structural motifs. The GA733-1 retroposon was localized either to chromosome region 1p32-1p31 or to 1p13-1q12, and the GA733-2 founder gene was localized to chromosome 4q.

A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.

Endothelial cell surfaces play key roles in several important physiological and pathological processes such as blood clotting, angiogenic responses, and inflammation. Here we describe the cloning and characterization of tie, a novel type of human endothelial cell surface receptor tyrosine kinase. The extracellular domain of the predicted tie protein product has an exceptional multidomain structure consisting of a cluster of three epidermal growth factor homology motifs embedded between two immunoglobulinlike loops, which are followed by three fibronectin type III repeats next to the transmembrane region. Additionally, a cDNA form lacking the first of the three epidermal growth factor homology domains was isolated, suggesting that alternative splicing creates different tie-type receptors. Cells transfected with tie cDNA expression vector produce glycosylated polypeptides of 117 kDa which are reactive to antisera raised against the tie carboxy terminus. The tie gene was located in chromosomal region 1p33 to 1p34. Expression of the tie gene appeared to be restricted in some cell lines; large amounts of tie mRNA were detected in endothelial cell lines and in some myeloid leukemia cell lines with erythroid and megakaryoblastoid characteristics. In addition, mRNA in situ studies further indicated the endothelial expression of the tie gene. The tie receptor tyrosine kinase may have evolved for multiple protein-protein interactions, possibly including cell adhesion to the vascular endothelium.

Wwox-Brca1 interaction: role in DNA repair pathway choice.

In this study, loss of expression of the fragile site-encoded Wwox protein was found to contribute to radiation and cisplatin resistance of cells, responses that could be associated with cancer recurrence and poor outcome. WWOX gene deletions occur in a variety of human cancer types, and reduced Wwox protein expression can be detected early during cancer development. We found that Wwox loss is followed by mild chromosome instability in genomes of mouse embryo fibroblast cells from Wwox-knockout mice. Human and mouse cells deficient for Wwox also exhibit significantly enhanced survival of ionizing radiation and bleomycin treatment, agents that induce double-strand breaks (DSBs). Cancer cells that survive radiation recur more rapidly in a xenograft model of irradiated breast cancer cells; Wwox-deficient cells exhibited significantly shorter tumor latencies vs Wwox-expressing cells. This Wwox effect has important consequences in human disease: in a cohort of cancer patients treated with radiation, Wwox deficiency significantly correlated with shorter overall survival times. In examining mechanisms underlying Wwox-dependent survival differences, we found that Wwox-deficient cells exhibit enhanced homology directed repair (HDR) and decreased non-homologous end-joining (NHEJ) repair, suggesting that Wwox contributes to DNA DSB repair pathway choice. Upon silencing of Rad51, a protein critical for HDR, Wwox-deficient cells were resensitized to radiation. We also demonstrated interaction of Wwox with Brca1, a driver of HDR, and show via immunofluorescent detection of repair proteins at ionizing radiation-induced DNA damage foci that Wwox expression suppresses DSB repair at the end-resection step of HDR. We propose a genome caretaker function for WWOX, in which Brca1-Wwox interaction supports NHEJ as the dominant DSB repair pathway in Wwox-sufficient cells. Taken together, the experimental results suggest that reduced Wwox expression, a common occurrence in cancers, dysregulates DSB repair, enhancing efficiency of likely mutagenic repair, and enabling radiation and cisplatin treatment resistance.

Lineage-specific gene rearrangement/deletion: a nonconservative model.

For the lymphocytic descendants of the hematopoietic differentiation pathway, characteristic gene rearrangements result in deletions of significant portions of chromosome regions specifying lymphocyte-specific gene products on either or both chromosomes of each involved pair. Molecular mechanisms facilitating the rearrangement/deletion events have been elegantly and fruitfully elaborated in the years since the first documentation of their occurrence by Hozumi and Tonegawa (N. Hozumi and S. Tonegawa, Proc. Natl. Acad. Sci. USA, 73:3628-3632, 1976). Numerous genetic phenomena observed in experiments or the literature suggest to us that specific genome rearrangement/deletion may be characteristic of and necessary for many, perhaps all, differentiating lineages. Thus we propose that on the way to terminal differentiation, cells within a particular lineage must rearrange specific chromosome regions characteristic for that lineage in order to switch off or on, perhaps concomitantly, requisite genes for that differentiation program. Such rearrangements and their ensuing deletions may have been undetected cytogenetically because of small size and/or because normal terminally differentiated cells do not enter mitosis. However, footprints of these rearrangements, most likely in aberrant form, may be preserved in preneoplastic cells and cancer cells of various lineages which exhibit characteristic deletions. An understanding of the cellular recombinational machinery involved in normal physiological genome rearrangements such as we propose may clarify some puzzling aspects of current theories concerning retinoblastoma, Wilms' tumor, and other "deletion syndromes" and the role of parental genome imprinting [B. M. Cattanach and M. Kirk, Nature (Lond.), 315: 496-498, 1985; C. Sapienza et al., Nature (Lond.), 328: 251-254, 1987; D. Solter, Annu. Rev. Genet., 22: 127-146, 1988]. The recombinatorial activity, when inappropriately expressed in dividing cells (i.e., cells which should be terminally differentiated but are still cycling for various reasons) could be responsible for such diverse phenomena as large deletions; chromosomal translocations into commonly deleted regions; amplicons; apparent nonrandom chromosome integration of viral genomes such as hepatitis B, human papilloma virus, papovaviruses, and retroviruses; and the observation of fragile sites. It could explain why these various phenomena often involve the same restricted regions of the genome. Some clues and consequences integral to the proposal are discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

Early deregulation of the the p16ink4a-cyclin D1/cyclin-dependent kinase 4-retinoblastoma pathway in cell proliferation-driven esophageal tumorigenesis in zinc-deficient rats.

The p16ink4a-cyclin D1/cyclin-dependent kinase 4 (Cdk4)-retinoblastoma (Rb) pathway has emerged as a critical target in oncogenesis. The zinc-deficient (ZD), N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal cancer model provides a tool to study cell proliferation and cell cycle control in cancer initiation. Weanling rats were fed a ZD or zinc-sufficient (ZS) diet for 5 weeks, and then given a dose of NMBA. After 14 weeks, esophageal tumor incidence was 88% in ZD rats with highly proliferative esophagi versus 0% in ZS rats. Expression of p16ink4a, cyclin D1, Cdk4, and Rb in relation to that of proliferating cell nuclear antigen was characterized in esophagi by immunohistochemistry at 0, 24, and 48 h, and 1, 3, 7, 10, and 14 weeks after NMBA treatment. As early as 24 h, proliferating cell nuclear antigen-positive focal hyperplastic lesions were detected in the suprabasal layers of ZD esophagi. At the same time, overexpression of cyclin D1, Cdk4, and Rb was found in the corresponding lesion in adjacent esophageal sections. By contrast, p16ink4a expression was reduced or absent. At all time points, p16ink4a showed reduced nuclear staining in ZD esophagi compared with that in ZS esophagi. In addition, increased expression of the hyperphosphorylated forms of Rb was detected in ZD esophagi by immunoblotting. Importantly, tumors were consistently observed in ZD esophagi at very early time points. These data, obtained using a unique in vivo model for esophageal cancer with rapid tumor induction, provide strong evidence for a link between deregulation of the p16ink4a-cyclin D1/Cdk4-Rb pathway and the initiation of esophageal tumors.