Butyric acid induces apoptosis by up-regulating Bax expression via stimulation of the c-Jun N-terminal kinase/activation protein-1 pathway in human colon cancer cells.

BACKGROUND & AIMS: The colonic epithelial cells near the top of the crypt have been shown to undergo apoptosis. Because butyric acid (BA) is the major short-chain fatty acid produced by fermentation of dietary fiber in the large bowel, it may be an important regulator of apoptosis in colorectal cancer. We investigated which signaling pathway is triggered by BA to undergo apoptosis in human colorectal cancer cells. METHODS: Human DiFi and FET colorectal cells were treated with BA to undergo apoptosis and were assayed for activation of c-Jun N-terminal kinase (JNK), transcription factor activation protein 1 (AP1) and NF-kappaB, and the proapoptotic molecule Bax. The contribution of specific pathways was assessed by examining the effects of dominant-negative mutants of JNK/AP1 or NF-kappaB on BA-induced Bax expression and apoptosis. RESULTS: BA-mediated DNA fragmentation and Bax induction were preceded by early stimulation of JNK, and the DNA-binding activities of AP1 and NF-kappaB. BA-induced enhancement of DNA fragmentation and stimulation of Bax promoter activity were blocked by the expression of dominant-negative mutants of JNK1 or AP1 but not NF-kappaB. CONCLUSIONS: These findings suggest that apoptosis triggered by BA involves transcriptional stimulation of the Bax gene via activation of the JNK/AP1 pathway in colonic epithelial cells.

Crossveinless 2 contains cysteine-rich domains and is required for high levels of BMP-like activity during the formation of the cross veins in Drosophila.

The BMP-like signaling mediated by the ligands Dpp and Gbb is required to reinforce the development of most veins in the Drosophila wing. However, the formation of the cross veins is especially sensitive to reductions in BMP-like signaling. We show here that the formation of the definitive cross veins occurs after the initial specification of the longitudinal veins in a process that requires localized BMP-like activity. Since Dpp and Gbb levels are not detectably higher in the early phases of cross vein development, other factors apparently account for this localized activity. Our evidence suggests that the product of the crossveinless 2 gene is a novel member of the BMP-like signaling pathway required to potentiate Gbb of Dpp signaling in the cross veins. crossveinless 2 is expressed at higher levels in the developing cross veins and is necessary for local BMP-like activity. The Crossveinless 2 protein contains a putative signal or transmembrane sequence, and a partial Von Willebrand Factor D domain similar to those known to regulate the formation of intramolecular and intermolecular bonds. It also contains five cysteine-rich domains, similar to the cysteine-rich domains found in Chordin, Short Gastrulation and Procollagen that are known to bind BMP-like ligands. These features strongly suggest that Crossveinless 2 acts extracelluarly or in the secretory pathway to directly potentiate Dpp or Gbb signaling.

Ectopic expression of wnt-5a in human renal cell carcinoma cells suppresses in vitro growth and telomerase activity.

Loss of genes located on chromosome 3p has been reported in many different types of human cancers, including renal cell carcinoma. Previous studies using a nontumorigenic human renal cell carcinoma cell line (RCC23) established from a stage III nonpapillary carcinoma with a loss of heterozygosity on 3p showed that microcell hybrids containing an introduced intact chromosome 3 resulted in a more differentiated phenotype including restored cellular senescence and repression of telomerase activity. Human wnt-5a has been cloned and mapped to chromosome 3p14-21. We have stably transfected human wnt-5a into RCC23 cells which results in in vitro growth suppression and repression of telomerase activity in a manner similar to that found in microcell hybrids containing an introduced intact chromosome 3.

Antisense wnt-5a mimics wnt-1-mediated C57MG mammary epithelial cell transformation.

The disruption of the normal expression of wnt-5a in cell lines and in tumors is becoming increasingly recognized as important in cell transformation and tumorigenesis. For example, in endometrial cancer wnt-5a is downregulated compared to normal tissue. Our laboratory has recently found that the ectopic expression of wnt-5a in human RCC23 renal carcinoma cells missing wnt-5a gene expression suppresses in vitro cell growth and telomerase enzyme activity. Furthermore, ectopic wnt-5a in MC-T16 uroepithelial cancer cells missing the region of chromosome 3p where wnt-5a has been mapped reverts uroepithelial cell tumorigenesis in athymic nude mice. These studies were based upon the previous finding that wnt-1 and wnt-2 transform C57MG mammary epithelial cells by downregulating the endogenous expression of wnt-5a. We now report that transfecting C57MG cells with a mammalian expression vector carrying antisense wnt-5a results in a cell phenotype that mimics cell transformation by ectopic wnt-1 or wnt-2. Correspondingly, wnt-1-transformed cells are partially reverted in the presence of ectopic wnt-5a. We conclude from this that wnt-5a is an important regulator of cell growth and differentiation and its loss of expression leads to cell transformation.

Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis.

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, delta hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.

Correlation of PCR-detected clonal gene rearrangements with bone marrow morphology in patients with B-lineage lymphomas.

Bone marrow biopsy is the conventional staging and posttherapy evaluation method for assessing marrow involvement by lymphoma. Polymerase chain reactions (PCR) for antigen receptor rearrangements have the potential to increase the detection of minimal degrees of marrow involvement. The present study is a concurrent morphologic and PCR evaluation of 225 staging or posttherapy marrow biopsies from 127 patients with B-lineage non-Hodgkin's lymphoma. The biopsies were morphologically categorized into four groups: group 1 (positive for lymphoma), 60 biopsies (27%); group 2 (suspicious for lymphoma), 20 biopsies (9%); group 3 (lymphocytic lesions of indeterminate biology), 22 biopsies (10%); and group 4 (negative for lymphoma), 123 biopsies (54%). Molecular studies were performed on concurrently obtained aspirates and used consensus immunoglobulin-heavy-chain (IgH) and IgH/bcl-2 gene PCR primers. A molecular clone was detected in 53 of the 225 aspirates (24%): group 1, 34 aspirates (57%); group 2, five aspirates (25%); group 3, one aspirate (5%); and group 4, 13 aspirates (11%). A PCR-positive aspirate was present in 47% of follicular lymphomas, 58% of diffuse large cell lymphomas, and 72% of the other lymphomas in the group I specimens. Morphology or PCR was positive in 79 of the 225 cases (35%). The molecular detection of clonality in the aspirate DNA from cases with positive morphologic findings was lower than anticipated. The discordance between morphology and PCR results may be related to sample variation between the trephine biopsy and aspirate, a failure to aspirate sufficient lymphoma cells, or insufficient primer homology for amplification. DNA extracted from trephine sections may provide results more concordant with morphology, because PCR detected a clone in 10 of 11 DNA specimens extracted from trephine biopsies with positive morphologic findings and PCR negative aspirates.

Reversion of uroepithelial cell tumorigenesis by the ectopic expression of human wnt-5a.

Wnt gene family members are thought to play an important role in cell growth and differentiation. When normal wnt gene expression is disrupted, there is the potential for cell transformation. We have found previously that a strong correlation exists between the loss of normal wnt-5a gene expression and cell transformation (Olson and Papkoff, Cell Growth & Differ., 5: 197-206, 1994). Recently, this has been tested directly using antisense wnt-5a, which, when expressed in mouse mammary cells, results in cell transformation (Olson and Gibo, Antisense wnt-5a transforms C57MG mouse mammary epithelial cells, manuscript in preparation). We hypothesize that wnt-5a is a growth-regulating gene, the disruption of which could result in tumorigenesis. The multistage progression of many human cancers involves the loss of normal tumor suppressor gene(s) activity. Several tumor suppressor genes are thought to map to chromosome 3p11-p25. We have studied the ectopic expression of human wnt-5a (3p14-p21) in a tumorigenic uroepithelial cell line with deletion of chromosome 3p13-p21.2. This results in loss of tumorigenicity in athymic nude mice and suppresses anchorage-independent cell growth in soft agar. This suggests that human wnt-5a is a novel tumor suppressor gene in uroepithelial cell carcinoma and may be one of the suppressor genes deleted or rearranged on chromosome 3p.

Percutaneous iodine-125 seed implantation for carcinoma of the prostate.

This study was performed to assess the early results of treating stages T1-T3 adenocarcinoma prostate with either Iodine-125 (125I) implant alone (Group 1), for smaller more well differentiated cancers, or with low dose external beam radiation followed by a 125I boost (XRT + 125I) (Group 2) for larger less well differentiated tumours. Eighty-six patients were followed for between 11 and 60 months with a mean follow up of 26.1. All patients were followed by regular prostate specific antigen (PSA) evaluations, and digital rectal examinations (DRE). Eighty patients had a follow-up biopsy at 1 year. Prostate specific antigen progression-free survival (PSA-PFS) was determined and defined. Complications and potency were also assessed. Early results of 125I prostate seed implantation are very promising especially for selected cases of localized carcinoma.

Performance of four seed-caching corvid species in operant tests of nonspatial and spatial memory.

The performance of 4 seed-caching corvid species was tested using 2 different operant nonmatching tasks. These species differ in their dependence on stored food, and differences in spatial memory tests have been correlated with better performance by the more cache-dependent species. Acquisition and retention of a color non-matching-to-sample task was tested in Experiment 1. Acquisition of the color task was not correlated with cache dependence, and no differences between species in performance during memory testing were found. Acquisition and retention of an operant spatial non-matching-to-sample task was tested in Experiment 2. Species differences in the spatial task were found for acquisition and during retention testing. The influence of natural history on the evolution of memory is discussed.

Detailed preliminary analysis of 125iodine implantation for localized prostate cancer using percutaneous approach.

Recent developments have permitted accurate seed placement and dosimetry for interstitial brachytherapy of selected patients with localized prostate cancer. We present our experience with 76 patients divided into 2 groups. Group 1 included 45 patients with smaller, more well differentiated tumors, usually less than 2 cm. in diameter on digital rectal examination or transrectal ultrasound and a Gleason score of less than 7 who were treated with 125iodine alone. Group 2 consisted of 31 patients with localized tumors greater than 2 cm. in diameter and/or a Gleason sum equal to or greater than 7 who were treated with low dose external beam radiation followed by 125iodine boost 4 weeks later. Complete clinical progression-free survival, including prostate specific antigen, digital rectal examination and biopsy, was 51% for group 1 and 63.3% for group 2, with a mean followup of 26.3 months. Prostate specific antigen progression-free survival was 97.7% for group 1 and 94.7% for group 2. These results appear to be superior to external beam radiation only although longer followup is needed to substantiate these favorable early results. The procedures were well tolerated with good potency sparing. They were performed on an outpatient or short stay basis and provided a good alternative to external beam radiation only or hormonal treatment for select patients with localized prostate cancer who may not be candidates for radical prostatectomy.

High-frequency toneburst-evoked ABR latency-intensity functions in sensorineural hearing-impaired humans.

The latency-intensity functions (LIFs) of ABRs elicited by high-frequency (8, 10, 12, and 14 kHz) toneburst stimuli were evaluated in 20 subjects with confirmed 'moderate' high-frequency sensorineural hearing loss. Wave V results from clicks and tonebursts revealed all intra- and intersession data to be reliable (p > 0.05). Linear regression curves were highly significant (p < or = 0.0001), indicating linear relationships for all stimuli analyzed. Comparisons between the linear regression curves from a previously reported normal-hearing subject group and this sensorineural hearing-impaired group showed no significant differences. This study demonstrated that tonebursts at 8, 10, and 12 kHz evoked ABRs which decreased in latency as a function of increasing intensity and that these LIFs were consistent and orderly (14 kHz was not determinable). These results will contribute information to facilitate the establishment of change criteria used to predict change in hearing during treatment with ototoxic medications.

Performance of four seed-caching corvid species in the radial-arm maze analog.

Four seed-caching corvid species were tested in an open-room analog of the radial-arm maze. During Experiment 1, the species more dependent on stored food. Clark's nutcrackers (Nucifraga columbiana) and pinyon jays (Gymnorhinus cyanocephalus), acquired the task more quickly and to higher accuracy levels than either scrub jays (Aphelocoma coerulescens) or Mexican jays (A. ultramarina). During Experiment 2, performance after retention intervals was tested. When intervals of 30-210 min were tested in ascending order, species differences observed during acquisition were again obtained. However, when intervals of 5-300 min were tested in random order, the species differed only at shorter intervals. During Experiment 3, only nutcrackers gave any indication of performing above chance after a 24-hr retention intervals. Results support the hypothesis of species differences in spatial information processing that correlate with dependence on stored food.

Regulated expression of Wnt family members during proliferation of C57mg mammary cells.

At least six members of the Wnt gene family are expressed in the murine mammary gland during growth and differentiation, whereas several other Wnt family members participate in malignant transformation of this tissue. We have used the C57mg mammary cell line, which naturally expresses the Wnt-4 and Wnt-5a genes, to examine Wnt gene expression during proliferation. The data show that the growth factors basic fibroblast growth factor, transforming growth factor beta 1, and epidermal growth factor are mitogenic for C57mg cells, and partial transformation by Wnt-1 can substitute for the proliferative signal provided by these factors. Several different mitogenic stimuli selectively down-modulate the levels of endogenous Wnt-4 and Wnt-5a RNA in C57mg cells. Partial transformation by either Wnt-1 or Wnt-2 is accompanied by a dramatic decrease in Wnt-4 RNA and a small decrease in Wnt-5a RNA. Mitogenic stimulation by basic fibroblast growth factor or partial transformation by Int-2, a fibroblast growth factor family member, also leads to a selective decrease in the levels of endogenous Wnt RNA. No expression of the Wnt-4 and Wnt-5a genes is detectable in C57mg cells that are fully transformed by the activated tyrosine kinase oncogene Neu. In contrast, overexpression of Wnt-5a in C57mg cells does not lead to a transformed phenotype and is not accompanied by a decrease in endogenous Wnt-4 RNA levels. Overexpression of Wnt-5a does lead to a small decrease in endogenous Wnt-5a levels, perhaps through autoregulation. These data indicate that Wnt-4 and Wnt-5a expression in mammary cells is responsive to growth regulatory signals, and the down-modulation of expression of either or both genes correlates with cell proliferation. The inverse correlation between expression of the endogenous Wnt genes and cell proliferation suggests that Wnt-4 and Wnt-5a may participate in restricting the proliferation of C57mg cells.

High-frequency monitoring for early detection of cisplatin ototoxicity.

Cisplatin can cause irreversible hearing loss initially detectable as impairment of high-frequency hearing with progression to lower frequencies. Many patients receiving cisplatin are too ill to tolerate lengthy audiometric testing. Therefore, a rapid and sensitive high-frequency monitoring strategy to detect cisplatin-induced ototoxicity is needed. Serial conventional (0.25 to 8 kHz) and high-frequency (> or = 8 kHz) threshold monitoring was performed in patients receiving cisplatin, resulting in 84% of ears showing hearing loss, of which 71% were detected first in frequencies of 8 kHz or greater. By analysis according to an individualized, specific high-frequency range, early identification of hearing loss occurred in 94% of ears showing change. This five-frequency procedure is a sensitive detector of ototoxicity and is proposed as an alternative monitoring protocol for patients receiving cisplatin who cannot tolerate extended testing.

Effects of response strategy and retention interval on performance of Clark's nutcrackers in a radial maze analogue.

Two groups of Clark's nutcrackers (Nucifraga columbiana) were trained to use either a stay or shift response strategy in a radial maze analogue. Each trial had a preretention stage, a retention interval, and a postretention test. In Experiment 1, acquisition with a 5-min retention interval was studied. Response strategy did not affect the rate at which the task was learned. Performance following longer retention intervals was tested in Experiments 2-4. Changes in retention intervals were presented in trial blocks of increasing duration in Experiment 2 and were randomly presented between trials in Experiment 3. Experiment 4 extended the retention interval to 24 hr. No difference in performance was found between the 2 groups in any of these experiments. These results suggest a flexible relationship between spatial memory and response requirement in food-hoarding birds for at least 1 spatial memory task.

High-frequency tone burst-evoked ABR latency-intensity functions.

High-frequency tone burst stimuli (8, 10, 12, and 14 kHz) have been developed and demonstrated to provide reliable and valid auditory brainstem responses (ABRs) in normal-hearing subjects. In this study, latency-intensity functions (LIFs) were determined using these stimuli in 14 normal-hearing individuals. Significant shifts in response latency occurred as a function of stimulus intensity for all tone burst frequencies. For each 10 dB shift in intensity, latency shifts for waves I and V were statistically significant except for one isolated instance. LIF slopes were comparable between frequencies, ranging from 0.020 to 0.030 msec/dB. These normal LIFs for high-frequency tone burst-evoked ABRs suggest the degree of response latency change that might be expected from, for example, progressive hearing loss due to ototoxic insult, although these phenomena may not be directly related.

High-frequency testing techniques and instrumentation for early detection of ototoxicity.

Veteran patients with certain types of infections and cancers are routinely treated with therapeutic agents having ototoxic potential, thus threatening loss of hearing sensitivity which preexists in the majority of these patients. To prevent communication deficits requiring intervention, this laboratory is developing instrumentation and techniques for early detection of ototoxicity. For this study, conventional (< or = 8 kHz) and high-frequency (> or = 8 kHz) hearing thresholds were monitored behaviorally in hospitalized veterans receiving treatment with ototoxic drugs. Data analysis revealed that monitoring only the high-frequency range would have identified 67% of ears showing change. A five-frequency range of hearing, specific to each individual, was identified for its high sensitivity to early ototoxic change. Monitoring of only these five frequencies in each patient would have identified 82% of ears that showed behavioral change. Auditory brainstem responses (ABR) were obtained in a subgroup using clicks and high-frequency (8-14 kHz) tone bursts. ABR latency/morphology changes were observed in 95% of ears demonstrating behavioral change. High-frequency tone-burst-evoked ABRs alone would have identified 93% of initial changes. Monitoring of high-frequency audition using these techniques shows promise for early detection of ototoxicity with potential for prevention of hearing loss in frequencies essential for verbal communication.

Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement.

Wnt genes encode secreted glycoproteins, and, because of their homology with the Drosophila segment polarity gene wingless, are likely to play important roles as modulators of local intercellular signalling during embryonic development. Although little is known of the mechanisms by which Wnts signal in an autocrine or paracrine manner, it is increasingly clear that cells can respond rapidly to Wnt signals in the absence of transcription, and that these responses may include changes in cell adhesion and cell movement. We review recent evidence from studies on Xenopus laevis and other systems, which demonstrate that (1) a subset of Wnts modulate gap junctional permeability, which may be a reflection of changes in cadherin-mediated cell adhesion, (2) embryos express beta-catenin and plakoglobin, which are homologs of the armadillo gene products, known to be involved in the wingless signalling pathway, and known to be found at cell junctions, and (3) overexpression of specific Wnts in Xenopus embryos leads to clear changes in cell behavior and movement.

Dissecting Wnt signalling pathways and Wnt-sensitive developmental processes through transient misexpression analyses in embryos of Xenopus laevis.

We review evidence that Xenopus Wnts (Xwnts) have activities consistent with their hypothesized roles as secreted signalling factors involved in multiple developmental processes. Transient misexpression of different Xwnts has distinct effects upon early development, and upon the formation of tissues in UV-irradiated embryos. Misexpression of Xwnts also has distinct effects on the in vitro differentiation of blastula cap explants. Cellular responses to Xwnt signals include changes in gap junctional permeability, altered responsiveness to growth factors, and possibly changes in cell adhesion. Current data suggest that a maternal Xwnt- or noggin-like activity is involved in the Nieuwkoop center activity during mesoderm induction, that Xwnt-8 participates in a pathway of differentiation as ventral mesoderm, and that Xwnt-5A is a potential modulator of morphogenetic movements.

Early detection of ototoxicity using high-frequency, tone-burst-evoked auditory brainstem responses.

Subjects receiving treatment with ototoxic agents were evaluated concurrently with conventional and high-frequency (> or = 8 kHz) behavioral threshold measures and with ABR to click and to 8, 10, 12, and 14 kHz tone-burst stimuli. Behavioral threshold data revealed ototoxic change in 51 percent of ears evaluated. Of these ears demonstrating behavioral change, 90 percent revealed concurrent ABR changes. If only ABR monitoring with high-frequency tone-burst stimuli had been used, 87 percent of allears showing behavioral change would have been identified. Three fourths of these would have been identified from wave V responses, with 87 percent identified from the two highest frequencies tested for each individual. This research suggests that behavioral change is reflected accurately in the ABR, that high-frequency tone bursts will identify a majority of initial ototoxic changes, and that monitoring hearing with high-frequency, tone-burst-evoked ABRs during treatment with potentially ototoxic agents is significantly more effective than click-evoked ABRs for early detection of ototoxicity.