Caspase cleavage of the APC tumor suppressor and release of an amino-terminal domain is required for the transcription-independent function of APC in apoptosis.

The adenomatous polyposis coli (APC) tumor suppressor is inactivated by mutation in most colorectal tumors. APC is a component of the Wnt signaling pathway and is best known for its ability to downregulate beta-catenin and consequent effects on transcriptional regulation. Previous work demonstrated that APC accelerates apoptosis-associated caspase activity independently of transcription, and suggested novel tumor suppressor functions of APC. In this work, we have mapped the APC apoptosis-accelerating region to amino acids (aa) 1-760 by testing a series of non-overlapping APC segments. Interestingly, this segment corresponds to a stable group II caspase cleavage product of APC released during apoptosis that includes the amino-terminal aa1-777. Mutation of the APC aspartic acid residue at position 777 to an alanine completely abolished in vitro cleavage of APC by a recombinant group II caspase and rendered the full-length protein unable to accelerate apoptosis in vitro. A truncated APC protein associated with familial and sporadic colorectal cancer, also unable to accelerate apoptosis in vitro and in vivo, is resistant to group II caspase cleavage. These results demonstrate that cleavage of APC and the subsequent release of an amino-terminal segment are necessary for the transcription-independent mechanism of APC-mediated apoptosis.

Alternative splicing of the APC gene and its association with terminal differentiation.

The human tumor suppressor gene, APC, is composed of at least 21 exons, 7 of which are alternatively expressed. Sixteen APC transcripts that differ in their 5'-most regions and arise by the alternative inclusion of 6 of these exons have been identified by reverse transcription-PCR analysis of RNA prepared from human, mouse, and rat cell lines and tissues. Tissue-specific differences were observed in the expression of APC transcripts without exon 1, a coding region for a heptad repeat that supports APC homodimerization. Transcripts without exon 1 were observed at high levels in postmitotic, differentiated tissues and in two cell lines following the induction of differentiation. Sequence analysis of these novel open reading frames predicts APC proteins with different amino-terminal domains and therefore potentially different abilities to associate with other proteins. Our findings suggest that the alternative splicing of APC leads to alternative forms of APC proteins with potentially unique functions in growth control and differentiation.

Microsatellite instability in colorectal adenocarcinoma cell lines that have full-length adenomatous polyposis coli protein.

Almost 20% of colon cancers are characterized by genomic instability at simple repeated sequences. This instability is the result of a deficient DNA mismatch repair system. Sporadic, as well as hereditary carcinomas of the proximal colon display this effect. In this study, we examined colorectal adenocarcinoma cell lines, with or without wild-type adenomatous polyposis coli (APC) protein, for the presence of microsatellite instability. The three cell lines that maintained full-length APC protein also displayed the highest level of instability, suggesting a negative correlation between APC mutations and microsatellite instability. This data, in addition to other studies that show a negative correlation between microsatellite instability and mutations in p53 and K-ras, support the idea of a second pathway for colorectal cancer development.

Demonstration of promoter activity and alternative splicing in the region 5' to exon 1 of the APC gene.

Screening of fetal brain and fetal retina complementary DNA (cDNA) libraries and exon-connection experiments using brain cDNA have identified three exons 5' to exon 1 of the adenomatous polyposis coli gene. The exons are termed (from 5'-3') 0.3, 0.1, and 0.2; exons 0.1 and 0.2 are contiguous genomically. Library screening revealed alternatively spliced cDNAs containing the following combinations of 5'-exons: 0.3 + 1 + 2, 0.3 + 2, 0.1 + 0.2 + 1 + 2, and 0.1 + 1 + 2. Exon-connection experiments also identified these four forms in mRNAs from tissues and cultured cell lines, along with two additional forms, 0.1 + 0.2 + 2 and 0.1 + 2. The multiple splice forms may lead to proteins of differing activity; for example, products derived from cDNAs without exon 1 will lack most of a heptad-repeat domain that supports formation of homodimers. No mRNA species combining 0.3 with either 0.1 or 0.2 were identified. The existence of two apparently separate 5'-ends of APC suggests the possibility of two independent promoters. The genomic sequence adjacent to exon 0.3 confers promotor activity when cloned in a chloramphenicol acetyltransferase expression vector and transfected into a colon cancer cell line.

Microsatellite instability in aberrant crypt foci from human colons.

Aberrant crypt foci (ACF) are distinct microscopic lesions of the colon thought to be the earliest identifiable precursors of colon cancer. As precursors of colon cancer, ACF may contain mutations in genes that are altered early in colorectal tumorigenesis. Candidates for these genes include APC, K-Ras, and those of the DNA mismatch repair system. Some colon cancers with mutations in DNA mismatch repair genes are characterized by genomic instability at simple repeated sequences, also known as microsatellite instability. In this study, we analyzed 19 ACF (> or = 20 crypts/focus) and adjoining, microscopically normal colonic mucosa from 10 colon cancer patients for the presence of microsatellite instability. DNA from two ACF from two different patients displayed microsatellite instability. None of the DNA samples from normal mucosa displayed microsatellite instability. These observations support the role of ACF as a precursor to colon cancer and provide some evidence that mutations in DNA mismatch repair genes are early somatic events in colon cancer.

A rapid screening method to detect nonsense and frameshift mutations: identification of disease-causing APC alleles.

A functional screen for nonsense and frameshift mutations has been devised that allows genes of interest to be scanned in segments. This assay is based on the cloning of these segments in-frame with a colorimetric marker gene (lacZ) followed by screening for the level of functional activity from the marker polypeptide (beta-galactosidase). Individuals at risk for any one of a number of genetic diseases, in particular familial adenomatous polyposis coli (APC), can be quickly screened for chain-terminating mutations introduced by stops and frameshifts. At present, scanning of the APC gene for mutation requires significant effort because it is a large gene and most APC mutations are unique. Therefore, this assay offers a powerful option for the diagnosis of this and other genetic diseases, as well as great potential for the development of a similar rapid screen to detect APC mutations in colorectal adenomas and carcinomas.

Response of colon cancer cell lines to the introduction of APC, a colon-specific tumor suppressor gene.

The APC gene, mutations in which are responsible for the inherited colon cancer syndrome adenomatous polyposis coli (APC), is described as a tumor suppressor gene. A full-length, wild-type APC gene was introduced by transfection into three human colon carcinoma cell lines, each characterized for mutations at loci involved in colon tumor formation. The response of each cell line to the introduction of APC differed with the genotype of the cell line. Some of the cell clones derived from these transfections displayed altered morphologies; some showed suppression of tumorigenicity based on growth in soft agar and tumor formation in nude mice. One cell line, SW480, could not be stably transfected with the APC gene. These results provide the first direct evidence that the APC gene can alter the transformation properties of colon carcinoma cells.

Catheter failure rates and time course with epidural versus combined spinal-epidural analgesia in labor.

BACKGROUND: The combined spinal-epidural technique for labor analgesia has several advantages over the traditional epidural technique, including faster onset, greater maternal satisfaction, and decreased need for physician boluses. Proponents of the epidural technique criticize the combined spinal-epidural technique, arguing that the epidural catheter remains untested and thus may not be reliable if needed for surgical intervention. We compared failure rates and time of failure between techniques in our tertiary-care academic practice. METHODS: Data regarding failed catheters were collected from October 2012 to September 2014 as part of our Quality Assurance program. Failed catheters were defined as any catheter replaced after it was considered to be properly placed and then determined to be intravascular, one sided or resulting in poor maternal analgesia or anesthesia. RESULTS: A total of 5487 analgesics were performed (3980 combined spinal-epidural; 1507 epidural). Eighty-five combined spinal-epidural catheters (2.1%) and 59 epidural catheters (3.9%) were replaced during labor (P<0.001). Mean time to replacement was 512±422min and 354±300min for the combined spinal-epidural (n=80) and epidural (n=57) groups, respectively (P=0.02). Median time to replacement was 398 [IQR 131-578] min and 281 [IQR 186-767] min for combined spinal-epidural and epidural groups, respectively (P<0.0001). CONCLUSION: We were able to demonstrate that catheters placed using a combined spinal-epidural technique were less likely to fail during labor and that the time to detection of a failed catheter was significantly longer in the combined spinal-epidural group. Our findings validate the combined spinal-epidural technique as reliable for labor analgesia and tend to refute the theory of the untested catheter.

Novel protein isoforms of the APC tumor suppressor in neural tissue.

The conventional protein isoform of the APC tumor suppressor is 310 kD and is encoded by exons 1 - 15 of the APC gene. Other RNAs are expressed from the APC gene and include one form that contains an exon upstream of exon 1, designated BS, but this transcript does not include exon 1. This transcript recently has been shown to be enriched in non-dividing, terminally-differentiated cells (Santoro and Groden, 1997). To determine if the BS-containing transcript encoded an alternate APC protein isoform, we generated and affinity-purified a polyclonal antibody directed to protein sequence predicted by exon BS. The BS antibody labeled a band of approximately 300 kD on immunoblots of cerebral and cerebellar tissue from adult human, baboon, rat and mouse. These same tissue lysates also contained prominent BS-reactive proteins of 290 kD, 200 kD and 150 kD. Lysates from mitotically active cells did not contain these APC isoforms. To verify that BS-reactive proteins were APC isoforms, BS-immunoprecipitates were blotted and labeled with commercially available APC antibodies. All four high molecular weight BS-antibody-precipitated proteins were recognized by antibodies directed against epitopes encoded by APC exons 2 and 15. BS isoforms were not, however, labeled with antibodies to an epitope encoded by APC exon 1, consistent with the prediction that BS - APC isoforms lack the domain encoded by these sequences. Like conventional APC, at least one of the four BS-APC protein isoforms also interacts with beta-catenin. BS-APC isoforms that lack exon 1-encoded sequences are incapable of dimerization with the conventional form of APC, yet retain the ability to bind beta-catenin. Such isoforms are likely to be functionally distinct from the conventional APC protein.

Biology of the adenomatous polyposis coli tumor suppressor.

The adenomatous polyposis coli (APC) gene was first identified as the gene mutated in an inherited syndrome of colon cancer predisposition known as familial adenomatous polyposis coli (FAP). Mutation of APC is also found in 80% of all colorectal adenomas and carcinomas and is one of the earliest mutations in colon cancer progression. Similar to other tumor suppressor genes, both APC alleles are inactivated by mutation in colon tumors, resulting in the loss of full-length protein in tumor cells. The functional significance of altering APC is the dysregulation of several physiologic processes that govern colonic epithelial cell homeostasis, which include cell cycle progression, migration, differentiation, and apoptosis. Roles for APC in some of these processes are in large part attributable to its ability to regulate cytosolic levels of the signaling molecule beta-catenin and to affect the transcriptional profile in cells. This article summarizes numerous genetic, biochemical, and cell biologic studies on the mechanisms of APC-mediated tumor suppression. Mouse models of FAP, in which the APC gene has been genetically inactivated, have been particularly useful in testing therapeutic and chemopreventive strategies. These data have significant implications for colorectal cancer treatment approaches as well as for understanding other disease genes and cancers of other tissue types.

Maternally inherited nonsyndromic hearing loss.

In this study we characterized clinically and evaluated molecularly a large family with maternally inherited hearing impairment. Relatives were evaluated audiologically and clinically, the most likely pattern of inheritance was deduced, and molecular DNA analysis for the known mitochondrial mutations associated with hearing impairment was performed. Clinical examination of several relatives showed a normal general state of health, but in 14 of the members tested variable degrees of sensorineural hearing loss were noted. The pedigree was established and demonstrated a clear pattern of maternal inheritance, with 34 of 38 offspring of deaf mothers being hearing impaired, but none of 22 offspring of deaf fathers having any hearing impairment. Since by far the most likely explanation of such a maternal inheritance pattern is a mitochondrial mutation, molecular testing for the three known mitochondrial mutations, A1555G, A7445G, and Cins7472, was performed on 27 of the relatives. All of the individuals tested had the normal sequence at the sites tested. This family with nonsyndromic sensorineural hearing loss has an inheritance pattern strongly suggestive of a mitochondrial mutation. However, molecular testing for the three known mitochondrial mutations associated with nonsyndromic hearing impairment was negative, implying that additional molecular defects can lead to the same phenotype. The search for this novel molecular defect is underway.

Bilateral keratitis as a manifestation of Lyme disease.

An 11-year-old girl developed bilateral keratitis, which we believe was a manifestation of Lyme disease. She had had several attacks of Lyme arthritis and was twice treated with parenteral penicillin. The keratitis developed five years after the initial episode of Lyme arthritis at a time when there were no other manifestations of Lyme disease. It cleared completely in both eyes after topical corticosteroid therapy.

The development of a stress survey schedule for persons with autism and other developmental disabilities.

The Stress Survey Schedule is an instrument for measuring stress in the lives of persons with autism and other developmental disabilities. Development of the survey and analysis of the underlying measurement structure of the instrument is reported in three studies. Through the use of exploratory and confirmatory analysis procedures, eight dimensions of stress were identified: Anticipation/Uncertainty, Changes and Threats, Unpleasant Events, Pleasant Events, Sensory/Personal Contact, Food Related Activity, Social/Environmental Interactions, and Ritual Related Stress. These stress dimensions are highly relevant to the problems of autism and have not been addressed by other stress surveys. The information obtained from the Stress Survey can be used to plan for strategies to reduce the stress before it occurs or results in maladaptive behavior.

High frequency of a common Bloom syndrome Ashkenazi mutation among Jews of Polish origin.

Bloom syndrome (BS) is an autosomal recessive disorder characterized by small stature, immunodeficiency, chromosomal instability, and a predisposition to different types of cancer. Although extremely rare in the general population, BS is seen in about 1 in 48,000 Ashkenazi Jews. Mutation analysis of seven Ashkenazi BS probands has shown that all were homozygous for the same mutation in the BLM gene: 2281delATCTGAinsTAGATTC, also known as blmAsh. This finding, along with the increased incidence of BS among Ashkenazi Jews, suggests a founder effect for BS in this population. The purpose of this study was to determine the frequency of blmAsh mutation carriers in a randomly sampled Ashkenazi Jewish population in Israel. The initial study group included 1,613 Ashkenazi Jews who were referred for routine DNA screening tests (cystic fibrosis, Gaucher, Canavan, fragile X). None had a family history of BS. A group of 552 non-Ashkenazi Jews served as controls. Mutation analysis was performed by PCR amplification followed by analysis of a specific BstN1 restriction site, created by the blmAsh mutation. All positive carriers were confirmed by direct sequencing. Sixteen blmAsh carriers were detected among 1,613 Ashkenazi Jews (1 in 101), compared to none among 552 non-Ashkenazi individuals. In this study, Ashkenazi Jews of biparental Polish descent had a significantly higher proportion of the blmAsh mutation (1 in 37) compared to Ashkenazi Jews of non-Polish descent. These results provide further evidence that a founder effect is responsible for the increased incidence of Bloom syndrome among Ashkenazi Jews, particularly those of Polish descent.

Different expressivity of two adjacent mutations of the APC gene.

AIMS AND BACKGROUND: The phenotypic expression of different APC mutations in familial adenomatous polyposis (FAP) is variable: two to three variants of the disease have been defined based on the severity of colonic manifestations. Age of onset and number of polypectomies per person-year of post-surgical follow-up were compared in two FAP families with very close mutation sites in the APC gene, in order to ascertain mutation-specific variation of expressivity. FAMILIES AND APC MUTATIONS: Family A (5 patients) carried a newly characterized mutation, a four bp deletion at codon 843. Family B (5 patients) carried a previously identified mutation at codon 835. RESULTS: Mean age of onset was 49.7 years in family A and 30.5 years in family B; number of polypectomies per person-year of follow-up was 1.05 for family A and 10.1 for family B (P < 0.001). CONCLUSIONS: There is significant variation of expressivity (allelic heterogeneity) in FAP between two mutations separated by only eight codons, located at the 5' extremity of APC gene exon 15.

Procedures to increase social interaction among adolescents with autism: a multiple baseline analysis.

Isolate behavior, impaired social relationships and lack of conversational skills are behaviors which characterize persons described as autistic. These deficits interfere with their effective integration into school and social environments. This study of three subjects investigates the use of imagery procedures, specifically, covert reinforcement, to increase peer verbal initiations in adolescents who are labeled "autistic" and concomitantly looks at the demand characteristics of therapist "request". A multiple baseline analysis was used to assess the effects of individual training sessions on peer verbal initiations. The results demonstrate that verbal initiations can be changed in socially deficient adolescents labeled "autistic".

Effects of fenfluramine on the behavior of autistic individuals.

The present report, part of a national, multicenter study to investigate the effects of fenfluramine on autistic behavior, describes findings on four autistic children ranging in age from 7 to 20 years. Additional performance and parental observation measures apart from those of the multicenter study are included. Results of this study which indicated no significant side effects, a reduction in some deviant behaviors and an improvement in activity level/attention span, provide support for earlier reports. The possibility that fenfluramine's apparently positive effects might be to simply reduce inappropriate behaviors via lethargy was examined and not supported.