Effect of wnt-1 and related proteins on gap junctional communication in Xenopus embryos.

The proto-oncogene wnt-1 (previously referred to as int-1) is thought to be important in embryonic pattern formation although its mechanisms of action are unknown. Premature and increased expression of the Wnt-1 protein, achieved by injection of synthetic wnt-1 RNA into fertilized Xenopus eggs, enhanced gap junctional communication between ventral cells of the developing embryo. This result is consistent with the hypothesis that Wnt proteins activate a receptor-mediated signal transduction pathway and that gap junctional communication can be a target of this pathway. The effects of two Wnt-1-related proteins on gap junctional communication were also investigated: overexpression of Xwnt-8 increased gap junctional coupling in a manner similar to Wnt-1, whereas Xwnt-5A did not. These findings are consistent with the existence of multiple receptors for Wnt proteins.

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.

Species differences in spatial memory among Clark's nutcrackers, scrub jays, and pigeons.

An operant nonmatching to sample procedure was used to compare the spatial memory abilities of 3 avian species. A trial consisted of the presentation of a spatially defined sample, a delay interval, and a 2-choice test during which the correct location was the new location. A single spatial location served as the sample in Experiment 1. The delay interval was manipulated using a titration procedure. In Experiment 2, 1, 2, or 3 sequentially illuminated locations served as the sample. The delay was 1 of 4 predetermined intervals. In Experiment 3, sample presentation was the same as Experiment 2, but the delay interval was titrated. In all of the experiments, the performance of nutcrackers was consistently better than the performance of scrub jays and pigeons (Experiment 1) and was correlated with differences in their foraging ecology.

Characteristics of spatial memory in pigeons.

Pigeons learned a delayed-alteration (DA) task in a T-maze. Very few trials were needed prior to accurate performance. Similarly, after little training the birds performed accurately with delays of 8-16 min. End-of-delay cues, possibly provided by the experimenter, response-based cues, and intramaze cues were all experimentally examined and rejected as bases for the birds' performances; pigeons appear to rely on spatial (extramaze) cues. Long-delay performances were undisturbed by changes in delay-interval stimuli (illumination shifts and transportation around the laboratory). Finally, birds were shown to acquire DA so quickly because of a potent tendency to avoid recently visited locations (i.e., a preexisting "win-shift" tendency). Characteristics of pigeon spatial memory thus include temporal persistence, resistance to retroactive interference, and a win-shift bias. In these respects spatial memory of pigeons parallels spatial memory of rats.

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.

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.

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.

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.

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.

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.

Reliability and validity of high-frequency (8-20 kHz) thresholds obtained on a computer-based audiometer as compared to a documented laboratory system.

A Macintosh computer-based audiometer (Virtual 320) was evaluated for reliability and validity of high-frequency (8-20 kHz) thresholds by comparison with a well documented laboratory high-frequency evaluation system (PARVA-HF). High-frequency earphones originally provided with the V320 for high-frequency testing required modification to improve reliability in calibration and in subject threshold testing. Twenty normal-hearing adults were evaluated in the 8-20 kHz frequency range on both testing systems. Results of intrasubject multiple-session testing were evaluated to determine the reliability of high-frequency thresholds obtained. The V320 produced reliable results comparable to the PARVA-HF. Validity of high-frequency thresholds was inferred by comparing V320 responses to those obtained with the PARVA-HF. Comparable findings between systems imply validity of thresholds obtained with the V320. Conventional frequency (0.25-8 kHz) threshold evaluation with the V320 and a Grason-Stadler 1701 audiometer also yielded comparable results.

Reliability of evoked responses to high-frequency (8-14 kHz) tone bursts.

Instrumentation to evaluate the auditory brainstem response to high-frequency (8-14 kHz) tone bursts has been developed in the Auditory Research Laboratory, Portland, Oregon VA Medical Center. This system is intended to monitor the audition of patients receiving ototoxic drugs who are unresponsive to behavioral test procedures. The reliability of responses obtained with the high-frequency tone-burst system was studied in 30 normal ears. Intrasubject variability of intersession data from response waves I, III, and V to tone bursts of frequencies 8, 10, 12, and 14 kHz was not significantly different from click response variability. The results of this study demonstrate the reliability of the ABR to these high-frequency tone-burst stimuli. This technique may provide early identification of hearing loss in unresponsive subjects receiving treatment with potentially ototoxic agents, thus allowing alternative treatments to minimize or prevent communicative handicap.

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.

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.

Amyloidosis: a rational approach to diagnosis by intraoral biopsy.

Amyloidosis and its oral manifestations are briefly described. The techniques for antemortem tissue diagnosis of amyloidosis are reviewed and evaluated. In screening for amyloidosis, biopsy of specific oral lesions (when present) is recommended instead of the traditional biopsy of normal-appearing gingiva. This approach is illustrated with four cases. Although rectal biopsy is generally accepted as the screening technique of choice, it is suggested that intraoral biopsy be considered as an easier, safer, more comfortable, and possibly more fruitful technique. It is advocated that in cases of both documented and suspected amyloidosis both intraoral and rectal biopsies should be performed in order to help provide documentation of the most useful technique for routine screening.

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.

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.

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.

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.