Inactivation of the retinoblastoma gene yields a mouse model of malignant colorectal cancer.

The retinoblastoma gene (Rb) is mutated at significant frequency in various human epithelial tumors, including colorectal cancer, and is strongly associated with metastatic disease. However, sole inactivation of Rb in the mouse has so far failed to yield epithelial cancers. Here, we specifically inactivate Rb and/or p53 in the urogenital epithelium and the intestine. We find that the loss of both tumor suppressors is unable to yield tumors in the transitional epithelium lining the bladder, kidneys and ureters. Instead, these mice develop highly metastatic tumors of neuroendocrine, not epithelial, origin within the urogenital tract to give prostate cancer in the males and vaginal tumors in the females. Additionally, we discovered that the sole inactivation of Rb in the intestine was sufficient to induce formation of metastatic colorectal adenocarcinomas. These tumors closely mirror the human disease in regard to the age of onset, histological appearance, invasiveness and metastatic potential. Like most human colorectal carcinomas, our murine Rb-deficient tumors demonstrate genomic instability and they show activation of ß-catenin. Deregulation of the Wnt/ß-catenin pathway is specific to the intestinal tumors, as genomic instability but not activation of ß-catenin was observed in the neuroendocrine tumors. To date, attempts to generate genetically engineered mouse models of colorectal cancer tumors have yielded mostly cancer of the small intestine, which rarely occurs in humans. Our system provides the opportunity to accurately model and study colorectal cancer in the mouse via a single gene mutation.

Inhibition of pituitary tumors in Rb mutant chimeras through E2f4 loss reveals a key suppressive role for the pRB/E2F pathway in urothelium and ganglionic carcinogenesis.

The retinoblastoma protein pRB suppresses tumorigenesis largely through regulation of the E2F transcription factors. E2F4, the most abundant E2F protein, is thought to act in cooperation with pRB to restrain cell proliferation. In this study, we analyse how loss of E2f4 affects the tumorigenicity of pRB-deficient tissues. As Rb(-/-);E2f4(-/-) germline mice die in utero, we generated Rb(-/-);E2f4(-/-) chimeric animals to allow examination of adult tumor phenotypes. We found that loss of E2f4 had a differential effect on known Rb-associated neuroendocrine tumors. It did not affect thyroid and adrenal glands tumors but partially suppressed lung neuroendocrine hyperplasia. The most striking effect was in the pituitary where E2F4 loss delayed the development, and reduced the incidence, of Rb mutant tumors. This tumor suppression increased the longevity of the Rb(-/-);E2f4(-/-) chimeric animals allowing us to identify novel tumor types. We observed ganglionic neuroendocrine neoplasms, lesions not associated earlier with mutation of either Rb or E2f4. Moreover, a subset of the Rb(-/-);E2f4(-/-) chimeras developed either low- or high-grade carcinomas in the urothelium transitional epithelium supporting a key role for Rb in bladder cancer.

The human tumor suppressor arf interacts with spinophilin/neurabin II, a type 1 protein-phosphatase-binding protein.

The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16(INK4a) and p14(ARF) (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.

Suppression of Ras-mediated NIH3T3 transformation by p19ARF does not involve alterations of cell growth properties.

The INK4a gene, one of the most frequently disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p19ARF, that both block cell proliferation. p16INK4a is a component of the Rb regulatory pathway, while p19ARF has been functionally related to p53. Moreover, p16INK4a is inactivated in many human tumors, while it has been very recently reported that p19ARF null mice develop tumors early in life. We show here that p19ARF is able to inhibit the formation of G418-resistant colonies when transfected into human and mouse cell lines expressing wild-type p53, regardless of p16 status. Moreover its amino terminal domain encoded by exon 1beta is still sufficient to obtain the same effect. We have analysed the ability of p19ARF to interfere with Ras-mediated cellular transformation in the NIH3T3 cell line. Cotransfection of p19ARF together with activated ras potently inhibited the formation of transformed foci in a dose-dependent manner. We have also isolated stable NIH3T3 transfectants expressing p19ARF and we have measured their growth properties as well as their efficiency of transformation by activated ras. Our results suggest that p19ARF can interfere with oncogene-mediated transformation, without significantly affecting NIH3T3 cell growth, at least at the levels of expression achieved in these experiments.

Characterization and genomic mapping of chimeric ERV9 endogenous retroviruses-host gene transcripts.

ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We performed Northern blot experiments using sequences from ERV9-LTR, and we observed a different pattern of expression in several different hemopoietic tumor cell lines. It is possible that by the result of a somatic integration event, or by virtue of their original dispersal in the genome, ERV9-LTRs may specifically induce the expression of different cellular sequences in different cell lineages. Here, we describe the identification and analysis of four chimeric cDNA clones isolated from the T-lymphoma Peer cell line, having a structure consistent with transcription initiation from an ERV9-LTR. All the cDNA clones represent transcripts derived from unique cellular sequences. We also report the genomic localization of these cDNA clones.

Mobilization of an ERV9 human endogenous retroviral element during primate evolution.

ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We report here the structural organization in different primate species of a zinc-finger coding gene whose expression is driven in humans by a solitary ERV9-LTR promoter. Using a PCR strategy and library screening, we were able to trace the origin of the insertion event in the primate lineage and to evaluate the impact of this event on gene structure. Our findings indicate that the integration of the ERV9 element occurred after the split of orangutang from the great apes, but before the divergence of the gorilla lineage. These results suggest that ERV9 elements have been mobile within the primate lineages and may still be active in humans.

High serum levels of angiotensin-converting enzyme in untreated Addison's disease.

Serum angiotensin-converting enzyme (ACE) in four patients with untreated Addison's disease was significantly higher [79.7 +/- 17.5 (SD) nmol min-1 ml-1] than in normal subjects (37.7 +/- 8.9 nmol min-1 ml-1, P less than 0.02). During corticosteroid hormone replacement therapy the enzyme levels returned within the normal range (44.0 +/- 7.6 nmol min-1 ml-1). In two additional patients the enzyme was assayed only while they were receiving therapy and found normal (38 and 52 nmol min-1 ml-1, respectively). In one of them, an increase ACE level (70 nmol min-1 ml-1) was found after therapy was reduced by 50%. Primary adrenal insufficiency is another disease in which ACE levels may be increased.

Habituation and sensitization of startle reflexes elicited electrically from the brainstem.

Repetitive elicitation of startle-like responses by electrical stimulation of the cochlear nucleus led to sensitization followed by habituation. In contrast, repetitive elicitation of startle-like responses by electrical stimulation of the reticular formation led only to sensitization. Since these different locations represent different points along the acoustic startle circuit, the data suggest that sensitization may be related to the motor side of reflex arcs, whereas habituation may be related to the sensory side.

Ontogeny of control over the acoustic startle reflex by visual prestimulation in the rat.

In Experiment I, rats 13-35 days of age were presented with acoustic startle stimuli in conjunction with light flashes or noise bursts at 4-320 msec lead times. Inhibition of the startle reflex by leading noise bursts was present on Days 13-15, whereas light flashes were not effective until Days 21-23. In Experiment II, 3 intensities of noise and 3 intensities of light preceded the reflex-eliciting stimuli on Days 16 and 35. All intensities of noise were effective on Day 16, whereas no visual stimuli were. On Day 35, all stimuli were effective. These data reveal that intrinsic visual structures and/or connections between these structures and reflex-control mechanisms mature relatively late in the rat.

Prenatal exposure to diazepam alters behavioral development in rats.

Characteristic potentiation of rat locomotion responses and acoustic startle reflexes that normally appear in the third postnatal week was absent in rats exposed to diazepam during the third week of gestation. Loss of these behaviors suggests a long-term effect that may result from changes in cellular development. Tissue undergoing neuronal differentation may be especially sensitive to drugs that act on the central nervous system, and the period in which differentiation occurs is perhaps critical for the induction of changes that are later expressed as altered behavior.

Development of the acoustic startle response in the rat: ontogenetic changes in the magnitude of inhibition by prepulse stimulation.

Three experiments examined the development of the acoustic startle reflex and its modification by a preliminary stimulus in the infant rat during the 2nd and 3rd postnatal weeks. The 1st experiment employed a white noise S1 (20 msec, 70 dB), the 2nd a cutaneous S1 (.5 msec, .5 mA and 1.0 mA shock), and the 3rd identical S1-S2 pairs (20 msec, 10 kHz, 110 dB tones). The results demonstrate a similar maturation of the prepulse modification pattern over days in the 3 experiments, evidenced mainly in the growth of inhibition. The findings indicate peripheral and central mechanisms that are maturing during the period of life under observation and that contribute to the developmental patterns of modification.