The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

IFN-gamma overexpression within the pancreas is not sufficient to rescue Pax4, Pax6, and Pdx-1 mutant mice from death.

In the presence of interferon-gamma (IFN-gamma), pancreatic ductal epithelial cells grow continuously, and islets undergo neogenesis. To determine whether these new islets are derived from conventional precursors, we tested whether IFN-gamma can complement the loss of transcription factors known to regulate pancreatic development. We analyzed the effect of a transgene on lethality in mice lacking the transcription factors Pax4, Pax6, or Pdx-1, by intercrossing such mice with transgenic mice whose pancreatic cells make IFN-gamma (ins-IFN-gamma mice). However, IFN-gamma expression did not rescue these mice from the lethal mutations, because no homozygous knockout mice carrying the IFN-gamma transgene survived, despite the survival of all other hemizygous gene combinations. This outcome demonstrates that the pathway for IFN-gamma regeneration requires the participation of Pax4, Pax6, and Pdx-1. We conclude that the striking islet regeneration observed in the ins-IFN-gamma NOD strain is regulated by the same transcription factors that control initial pancreatic development.

Hepatocyte migration during liver development requires Prox1.

Several genes are required during the early phases of liver specification, proliferation and differentiation. Here we report that Prox1 is required for hepatocyte migration. Loss of Prox1 leads to formation of a smaller liver with a reduced population of clustered hepatocytes surrounded by a laminin-rich basal membrane.

Pax 4 and 6 regulate gastrointestinal endocrine cell development.

The mechanisms behind the cell-specific and compartmentalized expression of gut and pancreatic hormones is largely unknown. We hereby report that deletion of the Pax 4 gene virtually eliminates duodenal and jejunal hormone-secreting cells, as well as serotonin and somatostatin cells of the distal stomach, while deletion of the Pax 6 gene eliminates duodenal GIP cells as well as gastrin and somatostatin cells of the distal stomach. Thus, together, these two genes regulate the differentiation of all proximal gastrointestinal endocrine cells and reflect common pathways for pancreatic and gastrointestinal endocrine cell differentiation.

Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas.

The functional unit of the endocrine pancreas is the islet of Langerhans. Islets are nested within the exocrine tissue of the pancreas and are composed of alpha-, beta-, delta- and gamma-cells. beta-Cells produce insulin and form the core of the islet, whereas alpha-, delta- and gamma-cells are arranged at the periphery of the islet and secrete glucagon, somatostatin and a pancreatic polypeptide, respectively. Little is known about the molecular and genetic factors regulating the lineage of the different endocrine cells. Pancreas development is known to be abolished in Pdx1-mutant mice and Pax4 mutants lack insulin-producing beta-cells. Here we show that the paired-box gene Pax6 is expressed during the early stages of pancreatic development and in mature endocrine cells. The pancreas of Pax6 homozygous mutant mice lack glucagon-producing cells, suggesting that Pax6 is essential for the differentiation of alpha-cells. As mice lacking Pax4 and Pax6 fail to develop any mature endocrine cells, we conclude that both Pax genes are required for endocrine fate in the pancreas.

The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas.

The mammalian pancreas contains two distinct cell populations: endocrine cells which secrete hormones into the bloodstream, and exocrine cells, which secrete enzymes into the digestive tract. The four endocrine cell types found in the adult pancreas-(alpha, beta, delta and PP-synthesize glucagon, insulin, somatostatin and pancreatic polypeptide, respectively. All of these endocrine cells arise from common multipotent precursors, which coexpress several hormones when they start to differentiate. Expression of some homeobox genes in the early developing pancreas has been reported. The Pax4 gene is expressed in the early pancreas, but is later restricted to beta cells. Inactivation of Pax4 by homologous recombination results in the absence of mature insulin- and somatostatin-producing cells (beta and delta, respectively) in the pancreas of Pax4 homozygous mutant mice, but glucagon-producing alpha cells are present in considerably higher numbers. We propose that the early expression of Pax4 in a subset of endocrine progenitors is essential for the differentiation of the beta and delta cell lineages. A default pathway would explain the elevated number of alpha cells in the absence of Pax4.

Prox 1, a prospero-related homeobox gene expressed during mouse development.

Prox 1, a likely mouse homologue of the Drosophila homeobox gene prospero has been cloned and its expression pattern analyzed during development. In Drosophila, prospero is expressed in the developing CNS, lens-secreting cone cells of the eye, and midgut. In the mouse, Prox 1 is expressed in many of the same tissues: young neurons of the subventricular region of the CNS, developing eye lens and pancreas. Expression is also detected in the developing liver and heart, as well as transiently in the skeletal muscles. The similarities in protein sequence and expression patterns between the mouse and fly cognate genes suggest that Prox 1 may play, among others, a fundamental role in early development of the murine CNS.

Homeoboxes in flatworms.

A search for homeobox-containing genes was done in the genome of a primitive metazoan, the parasitic tapeworm Echinococcus granulosus. Five different homeoboxes were isolated, none of them belonging to the classical Antennapedia-type. Three of the homeodomains are similar to those from the Drosophila melanogaster NK-type genes. The fourth homeodomain shares extensive identity with that of the recently reported homeobox-containing gene goosecoid from Xenopus laevis. The third helix (the recognition helix) of the fifth isolated homeodomain is identical to that of the Xlim-1 gene of X. laevis and the lin11 gene of Caenorharbditis elegans. Using PCR, some Antennapedia-type homeoboxes were cloned from the genome of two other Platyhelminthes, Dugesia tigrina (planaria) and Fasciola hepatica. These data suggest that, contrary to what is found for the majority of the more complex metazoans, Platyhelminthes contain few homeobox genes belonging to the Antennapedia-type.

Cloning and characterization of cDNA encoding canine alpha-L-iduronidase. mRNA deficiency in mucopolysaccharidosis I dog.

alpha-L-Iduronidase is a lysosomal enzyme, the deficiency of which causes mucopolysaccharidosis I (MPS I); a canine MPS I colony has been bred to test therapeutic intervention. The enzyme was purified to apparent homogeneity from canine testis and found to consist of two electrophoretically separable proteins that had common internal peptides but differed at their amino termini. A 57-base oligonucleotide, corresponding to the most probable codons of the longest peptide, was used to screen a canine testis cDNA library. Three cDNAs were isolated, two of which lacked the 5'-end whereas the third was full-length except for a small internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The amino terminus of the larger protein, glutamic acid 26, is at the predicted signal peptide cleavage site, whereas the amino terminus of the smaller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of GenBank showed that the amino acid sequence of alpha-L-iduronidase has similarity to that of a bacterial beta-xylosidase. A full-length cDNA corresponding to the composite sequence was constructed (pcIdu) and inserted into the pSVL expression vector (pSVcIdu). Two days after Cos-1 cells were transfected with pSVcIdu, their intracellular and secreted level of alpha-L-iduronidase activity has increased 8- and 22-fold, respectively, over the endogenous activity. Fibroblasts of MPS I dogs, which have no alpha-L-iduronidase activity, lacked the normal alpha-L-iduronidase mRNA of 2.2 kilobases and contained instead a trace amount of a 2.8-kilobase species. Isolation and characterization of an expressible alpha-L-iduronidase cDNA represents the first step toward mutation analysis and replacement therapy.

Apparent generation of a segmented mRNA from two separate tandem gene families in Trypanosoma cruzi.

Using a cDNA for an abundant Trypanosoma cruzi mRNA as probe, we have cloned and sequenced a gene which is organized in at least 20 nearly perfect tandem repeats of 940 base pairs. The 5' end of the mRNA has been sequenced by primer extension and found to contain a 35 nucleotide mini-exon (or spliced-leader) sequence that is ubiquitous in trypanosome mRNAs. This sequence, however, is not present in the tandem genomic repeats which encode the exon containing the major portion of the mRNA. Previous studies have shown that the 35-nucleotide sequence is encoded by a separate tandem gene family. One model to explain the formation of a segmented mRNA invokes priming of transcription by a small RNA which contains the leader sequence at its 5' end. However, northern blot analysis of total trypanosome RNA reveals a ladder of molecules larger than the mature mRNA, which appear to be faithful multimeric copies of the tandem gene. The discrete sizes of these RNAs correspond to those expected for partially processed precursors. These observations lend credence to the possibility of an alternative model where segmented mRNAs are generated by inter-molecular splicing.