Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms.

Familial cancer syndromes have helped to define the role of tumor suppressor genes in the development of cancer. The dominantly inherited Li-Fraumeni syndrome (LFS) is of particular interest because of the diversity of childhood and adult tumors that occur in affected individuals. The rarity and high mortality of LFS precluded formal linkage analysis. The alternative approach was to select the most plausible candidate gene. The tumor suppressor gene, p53, was studied because of previous indications that this gene is inactivated in the sporadic (nonfamilial) forms of most cancers that are associated with LFS. Germ line p53 mutations have been detected in all five LFS families analyzed. These mutations do not produce amounts of mutant p53 protein expected to exert a trans-dominant loss of function effect on wild-type p53 protein. The frequency of germ line p53 mutations can now be examined in additional families with LFS, and in other cancer patients and families with clinical features that might be attributed to the mutation.

p53 functions as a cell cycle control protein in osteosarcomas.

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

Equal transcription of wild-type and mutant p53 using bicistronic vectors results in the wild-type phenotype.

Somatic and germ-line mutations of p53 alleles inactivate the function of the protein. It has been suggested that mutant p53 can inactivate the wild-type protein and therefore have a trans-dominant negative effect. To investigate the interaction between wild-type and mutant proteins when both alleles are equally transcribed, we designed bicistronic vectors containing the internal ribosome entry site of the encephalomyocarditis virus and expressing wild-type and mutant p53. Analysis of the transcriptional activity and of the effect on cell growth of these plasmids indicates that the mutant protein is unable to completely suppress wild-type function. These results could explain why the inactivation of both p53 alleles is required in cancer development.

A functional screen for germ line p53 mutations based on transcriptional activation.

Germ line p53 mutations represent a genetic predisposition for cancer development. At the present time, their detection requires extensive work and their functional significance must be documented. Therefore, we have designed a simple biological assay which detects functionally significant germ line p53 mutations. This assay is based on the cloning of the patient's p53 complementary DNA into a eukaryotic expression vector followed by the cotransfection into human cells of the recombinant vector with a reporter plasmid for the transcriptional activity of p53. This assay potentially offers a powerful method to screen fibroblasts or lymphocytes from patients for germ line mutations which inactivate the p53 tumor suppressor gene.

Distribution and physical properties of BCA200, a Mr 200,000 glycoprotein selectively associated with human breast cancer.

Of 122 mouse monoclonal antibodies selective for human breast cancer, 13 immunoprecipitated an acidic glycoprotein from SK-Br-3 and ZR-75-30 human breast cancer cells. The antigen (BCA200) migrates with an apparent molecular weight of 200,000 on reducing and 180,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a single polypeptide chain with a folded domain stabilized by a disulfide bond. Cross-blocking and sandwich immunoassays detected at least three distinct antigenic determinants on BCA200. Scatchard experiments measured 1,000,000 to 5,000,000 antigen copies per SK-Br-3 cell. The tissue distribution of BCA200 was studied using two monoclonals to different epitopes. Neither antibody stained any cells in human blood. When frozen sections of 20 normal human tissues were immunoperoxidase stained, the only positive structures were mucinous glands of colon, transitional epithelium of bladder, sweat glands of skin, and acinar epithelium of breast. Antibody 454C11 stained 16 of 21 breast tumor frozen sections and 9 of 12 breast cancer cell lines, while antibody 520C9 stained 5 of 20 breast tumors and 4 of 10 breast cancer lines. Cross-reaction was observed with lung, prostatic, pancreatic, endometrial, and ovarian cancer, but not with lymphoma, melanoma, colon, stomach, bladder, or esophageal cancer. When conjugated to ricin A chain, 10 of 13 antibodies produced immunotoxins selectively cytotoxic to SK-Br-3 breast cancer cells.

A functional assay for heterozygous mutations in the GTPase activating protein related domain of the neurofibromatosis type 1 gene.

The GTPase-activating protein related domain of the human neurofibromatosis type 1 protein (NF1GRD) can down-regulate RAS in Saccharomyces cerevisiae. Using a technique termed the FASAY method, for Functional Analysis of Separated Alleles in Yeast, we designed a rapid method for detection of heterozygous NF1GRD loss-of-function mutations. In our method, PCR amplified NF1GRD cDNA is directly cloned into a centromeric vector by homologous recombination in a cdc25 temperature-sensitive mutant strain expressing human Ha-ras. This strain is dependent on the Ha-ras for growth, allowing a simple growth assay for NF1GRD loss-of-function mutations. In a test of our method, two alternatively spliced NF1GRD cDNAs (type I and II) inhibited yeast growth whereas four mutants with amino acid substitutions at highly conserved residues did not. This simple method thus permits the rapid screening for heterozygous germline or somatic NF1GRD mutations. In an initial application of this method, no mutations disrupting NF1GRD function were detected in lymphoblasts from 11 previously untested neurofibromatosis type 1 patients.

Variability in tactile projection patterns to cerebellar folia crus IIA of the Norway rat.

Tactile responses in the granule cell layers of the cerebellar hemispheres of rats are topographically arranged as a series of patches each representing a different region of the body surface. Previous observations had suggested that patches representing specific body parts recur in similar folial positions in different individuals; however, these relationships were not quantified. In this study we make inter-animal comparisons of the detailed distribution of receptive fields in the granule cell layer of the crown of crus IIa by using physiological mapping techniques. The results suggest that maps from different individuals do, in fact, share several topological features. These include the regions of the body surface represented, the general proportions of these representations, the relative positions of patches representing the same body parts, and the organization of receptive fields within patches located in similar positions. The principal variability seen in these comparisons was in the detailed neighborhood relations between different patches. As a result of the analysis of the consistent and variable features of these maps, we propose and discuss a new role for these cerebellar regions in coordinating the acquisition of tactile sensory information.

Fractured cutaneous projections to the granule cell layer of the posterior cerebellar hemisphere of the domestic cat.

Snider 's pioneering studies of tactile responses in the cerebellar cortex of cats and monkeys suggested that posterior regions of the cerebellar hemispheres receive somatotopically organized projections. However, recent studies in rats, using high-density, in depth microelectrode mapping methods, have shown that tactile projections to the granule cell layer of the cerebellar hemispheres are somatotopically disrupted. We reexamined the organization of cutaneous projections to cerebellar hemispheric cortex in cats by using micromapping methods. Natural stimulation of cutaneous surfaces evokes short-latency (mossy-fiber-induced) multiple unit responses in the cerebellar granule cell layer of crus II and paramedian lobule in both ketamine- and barbiturate-anesthetized cats. Facial structures are represented in several of the most caudomedial folia of crus II as well as in three of the rostral folia of the paramedian lobule. In several of these paramedian folia, facial projections are interspersed with projections from the forelimb. Forelimb structures alone are represented in two intermediate folia of the paramedian lobule. No cutaneous projections were found from the trunk or hindlimb. All projections were from ipsilateral receptive fields. In four folia of crus II and six folia of the paramedian lobule, cutaneous projections form a mosaic of patchlike projections. Within single patches, projections are somatotopically organized, but projections to adjacent patches come from noncontiguous body regions. Within a single folium , a particular facial region may be represented in two or three spatially separated patches. Facial patches are small, usually less than 1 mm2. Forelimb patches are usually larger, often extending the full length and breadth of a folial crown. Patches with like receptive fields are not organized in zonal sagittal strips. Rather than being somatotopically organized, cutaneous mossy fiber projections to granule cells in cats, as in rats, reveal a more complex mosaic pattern of organization.

Nicotine elimination and tolerance in non-dependent cigarette smokers.

Although most smokers are nicotine-dependent, recent studies suggest that some very light smokers ("chippers", who smoke fewer than five cigarettes per day) may smoke for decades without developing dependence. It was considered that slowed nicotine elimination and/or reduced nicotine tolerance might underlie chippers' ability to maintain smoking at such low levels. To evaluate this hypothesis, we studied the elimination kinetics and pharmacodynamics of nicotine in chippers and matched regular smokers. Plasma nicotine levels and cardiovascular responses were observed for several hours after subjects were administered uniform doses of tobacco smoke. Chippers did show less chronic nicotine tolerance, but only on some response measures. Their rates of nicotine elimination equaled those of regular smokers. This finding, when coupled with other data about chippers' smoking patterns and nicotine absorption, establish that chippers cannot maintain substantial plasma nicotine levels between cigarettes, and thus suggest that attempts to maintain minimal trough levels of nicotine do not underlie chippers' smoking.

Superior colliculus projections to tactile areas of rat cerebellar hemispheres.

The spatial organization of the projections of the superior colliculus (SC) to the contralateral cerebellar hemisphere and posterior vermis was examined using micromapping techniques in barbiturate or ketamine anesthetized rats. Electrical stimulation of the tactile-responsive intermediate layers of SC evoked responses in tactile regions of the contralateral granule cell (GC) layer of cerebellar cortex. In most experiments, the SC stimulating electrode remained stationary while the cerebellar electrode mapped the region of the GC layer activated by SC stimulation. In a few experiments, the cerebellar recording electrode remained fixed while the SC electrode mapped the area of effective stimulation. Puncture densities up to 60/sq.mm were necessary to fully determine the spatial pattern of these projections. Major results are: (1) cerebellar responses to SC stimulation are found in the GC layer of contralateral crus I, crus II, lobulus simplex, and the paramedian lobule; no responses were seen in the uvula. The anterior lobe was not examined; (2) peripheral receptive fields (RFs) of the interconnected SC and GC loci consist exclusively of facial structures (especially vibrissae, crown, bridge of nose, and eyelid); (3) SC sources project within the confines of GC layer patches with similar RFs; (4) GC layer responses typically consist of an early, low amplitude potential, followed by a multiple-unit response; and (5) latencies of GC layer responses to SC stimulation are 2.2-2.5 msec for the early potential, and 2.5-3.5 msec for the multiple-unit response. This is the first physiological demonstration of projections from the superior colliculus to the cerebellar hemispheres. The results of this and other studies in our laboratory indicate that the tactile areas of the cerebellar hemispheres receive convergent inputs from peripheral, tectal, and SI cortical tactile sources.

Somatotopic organization of SI corticotectal projections in rats.

The somatotopic organization of somatosensory (SI) cortical projections to the deep layers of the superior colliculus (SC) in rats was examined using neurophysiological micromapping methods. Electrical stimulation of SI produced SC unit responses at the same depths as did peripheral tactile stimulation, in SC laminae IV and VI; mapping of corticotectal projections was performed only in lamina IV. Antidromic activation of the pathway revealed that corticotectal cell bodies are located in SI layer V. Corticotectal projections were found to interconnect only those SI and collicular areas which have similar tactile receptive fields.

Trigeminocerebellar mossy fiber branching to granule cell layer patches in the rat cerebellum.

Recent micromapping studies of tactile areas in rat cerebellum have revealed a spatially precise mosaic of granule cell layer areas termed 'patches', with multiple patches on several folia resulting in multiple representations of the same body part. The research reported in the present paper tested the hypothesis that trigeminocerebellar mossy fiber branching is patch-related, so that collaterals terminate only within the boundaries of one or more of the tiny (often less than 1 sq. mm) patches having similar receptive fields. This hypothesis was supported by 3 separate series of electrophysiological experiments: (1) electrical stimulation in a patch resulted in short latency, low threshold responses confined within the boundaries of a second patch having a similar receptive field; (2) antidromic collison tests demonstrated that collaterals of axons originating in nucleus interpolaris reach pairs of patches with similar receptive fields; and (3) micromapping of the area from which a particular interpolaris neuron could be antidromically activated revealed that local terminal branching of a given mossy fiber axon appeared to be confined within the boundaries of a single patch. Together these results indicate that trigeminocerebellar mossy fiber branching is related to the fractured somatotopical organization of the granule cell layer. Possible functional implications of such mossy fiber branching are discussed.

Attentional mediation of cigarette smoking's effect on anxiety.

It was hypothesized that smoking's calming effects are cognitively mediated and depend on the presence of a benign distractor, smoking narrows the focus of attention, thereby reducing anxiety by facilitating distraction from stressful cognitions (cf. C. M. Steele & R. A. Josephs's [1988] attention-allocation model of alcohol reinforcement). This notion was tested by examining the effect of smoking (vs. not smoking) on anxiety with and without a concurrent distraction in 82 smokers; distraction effects were also assessed in 42 nonsmokers. As predicted, smoking reduced anxiety only when paired with a distractor. Further, these findings could not be explained by direct nicotine effects or nicotine withdrawal. Several measures of attention allocation failed to confirm the hypothesized cognitive mechanisms, however. Implications of the findings are discussed.

Temptations to smoke after quitting: a comparison of lapsers and maintainers.

This study addresses whether characteristics of temptations to smoke differ for participants who quit smoking and maintain abstinence compared to those who quit and then lapse. Participants used hand-held computers to record temptations and were beeped at random for base-rate assessments. We used generalized estimating equations to compare 1,851 temptation episodes and 5,192 random assessments recorded by 151 participants (116 lapsers, 35 maintainers) over 1-23 days of abstinence. Compared to randomly sampled occasions, temptations were marked by greater negative affect, restlessness, attention disturbance, and exposure to smoking cues; participants were also more likely to be eating or drinking during temptations. Temptations reported by lapsers and maintainers did not differ in any respect, including their reported coping. The results highlight situational variance over individual differences.

Nicotine withdrawal in chippers and regular smokers: subjective and cognitive effects.

From previous studies, chippers (very light, long-time cigarette smokers) seem not to be nicotine dependent, despite decades of smoking. The effect of tobacco deprivation on chippers' withdrawal reactions was examined. Matched groups of 26 chippers and 25 regular smokers were studied while abstaining or smoking for 2-day blocks, with assessments administered 5 times daily by palm-top computers. As hypothesized, chippers showed no changes as a result of nicotine deprivation. In contrast, regular smokers demonstrated distinct changes in craving, mood, arousal, and sleep disturbance. The computers also tested participants' cognitive performance. Unlike chippers, regular smokers' performance on complex tasks was slower under deprivation; the effect could not be explained by changes in motor performance or simple reaction time. Results suggest performance may have been improved by nicotine rather than by worsened by withdrawal.

Smoking and attention: a review and reformulation of the stimulus-filter hypothesis.

An accumulation of evidence suggests that smoking may be reinforcing, in part, due to nicotine's capacity to enhance attentional processing. Correspondingly, the stimulus-filter model of nicotine reinforcement asserts that nicotine facilitates cognitive performance by acting as a stimulus-barrier, thereby screening irrelevant and annoying stimuli from the smoker's awareness. A review of the available data suggests that while nicotine does appear to reliably enhance sustained, divided, and focused attention, the stimulus-filter model falls short of adequately explaining the findings. An alternative, attention, allocation model of nicotine reinforcement is reviewed, the tenets of which suggest that nicotine differentially augments attentional processing via its propensity to: (a) induce attentional narrowing, and (b) increase perceptual processing capacity. The motivational implications of the model, including smokers' use of nicotine to dampen stress, are discussed.

First lapses to smoking: within-subjects analysis of real-time reports.

Studies of smoking relapse and temptation episodes have relied on retrospective recall and confounded between- and within-subject variability. Real-time data on temptations and lapses to smoke were gathered using palm-top computers. We made within-subject comparisons of the initial lapse, a temptation episode, and base rate data obtained through randomly scheduled assessments. Negative affect discriminated all three situations, with lapses worse than temptations, and temptations worse than random situations. Participants attributed lapses to negative mood and smoking cues, whereas temptations were more often attributed to behavioral transitions. Participants were 12 times more likely to report coping in temptations than in lapses. However, only cognitive (vs. behavioral) coping strategies were effective. Lapses (vs. the other situations) were more likely to occur when smoking was permitted, when cigarettes were easily available, and in the presence of other smokers. The results have clinical implications, and the computerized monitoring methods may be applicable to an array of clinical research problems.

Remember that? A comparison of real-time versus retrospective recall of smoking lapses.

Research and treatment assessments often rely on retrospective recall of events. The accuracy of recall was tested using accounts of smoking lapse episodes from 127 participants who had quit smoking, and lapses and temptations were recorded in near-real time using a hand-held computer. These computer records were compared with retrospective accounts elicited 12 weeks later, with a focus on recall of lapses in 4 content domains: mood, activity, episode Triggers, and abstinence violation effects. Recall of lapses was quite poor: Average kappas for items ranged from 0.18 to 0.27. Mean profile rs assessing recall for the overall pattern of behavior were .36, .30, .33, and .44 for these domains, respectively. In recall, participants overestimated their negative affect and the number of cigarettes they had smoked during the lapse, and their recall was influenced by current smoking status. The findings suggest caution in the use of recall in research and intervention.

Progression from a smoking lapse to relapse: prediction from abstinence violation effects, nicotine dependence, and lapse characteristics.

Determinants of progression from an initial smoking lapse to relapse, using prospective data from 133 participants were examined. Participants used palm-top computers to record their first lapse, and their reaction to it, within minutes of the event, and were followed for 3 months to assess subsequent smoking. Indicators of the Abstinence Violation Effect--self-efficacy, attributions, and affective reactions to the lapse--generally failed to predict progression to relapse, but participants who felt like giving up after the first lapse progressed more rapidly to a second lapse. Participants who attempted restorative coping were less likely to progress to another lapse on the same day. Those whose lapses were triggered by stress progressed more quickly, whereas those triggered by eating and drinking or accompanied by alcohol consumption progressed more slowly. More nicotine-dependent participants progressed more rapidly toward relapse, but neither the amount smoked in the first lapse nor its subjective reinforcement predicted progression.

Does smoking abstinence self-efficacy vary across situations? Identifying context-specificity within the Relapse Situation Efficacy Questionnaire.

According to self-efficacy and relapse theories. abstinence self-efficacy (ASE) ratings should be context-specific: they should vary across situations. This variability may be important, as it could signal high-risk for relapse situations. In this study, confirmatory factor analysis was used to identify situational variability in a novel ASE assessment (Relapse Situation Efficacy Questionnaire. or RSEQ). Results supported a hierarchical structure, where both context-specific and unidimensional measures of ASE exist within the assessment. Context-specific factors included Negative Affect, Positive Affect, Restrictive Situations (to smoking). Idle Time, Social-Food Situations, Low Arousal, and Craving. Multiple context-specific factors and the aggregate factor predicted cessation outcome, even after controlling for concurrent smoking rate. However, the context rated with the least confidence proved to be the best outcome predictor, suggesting the existence of "Achilles' heel" situations. These data indicate the internal and predictive validity of the RSEQ.