BRCA1 targets G2/M cell cycle proteins for ubiquitination and proteasomal degradation.

The BRCA1 tumor suppressor protein heterodimerizes with its partner protein, BARD1, via the RING domain present in both proteins. The heterodimer contains an E3 ubiquitin ligase activity and participates in multiple cellular functions such as cell cycle control, DNA repair and regulation of gene transcription, collectively aimed at maintaining genomic stability and tumor suppression. Yet, the precise role of BRCA1 E3 ligase in these cellular functions is poorly understood. We present data showing that BRCA1 ubiquitinates G2/M cell cycle proteins, cyclin B and Cdc25C, leading to their accelerated degradation via a mechanism that is independent of APC/C. BRCA1-dependent degradation of cyclin B and Cdc25C is reversed by proteasome inhibitors and is enhanced following DNA damage, which may represent a possible mechanism to prevent cyclin B and Cdc25C accumulation, a requirement for mitotic entry. Our data provide mechanistic insight into how BRCA1 E3 ligase activity regulates the G2/M cell cycle checkpoint and, thus, contributes to maintenance of genomic stability.

Insulin-like growth factor-I controls BRCA1 gene expression through activation of transcription factor Sp1.

The insulin-like growth factors (IGFs) have a central role in mammary gland growth and differentiation as well as in breast cancer development. The BRCA1 gene encodes a pleiotropic protein that functions as a transcription factor. Germline BRCA1 mutations are associated with inherited predisposition to breast and ovarian cancer and confer a substantially increased risk for developing these neoplasms. Several lines of evidence led us to hypothesize that there is a functional interaction between the BRCA1 and IGF-I systems relevant to breast cancer biology. The present study tested the notion that BRCA1 gene expression is regulated by the IGF-I signaling pathway. Results of Western immunoblotting and RT-PCR analyses show that IGF-I stimulates BRCA1 protein and mRNA levels. Transient transfection experiments using BRCA1 promoter-luciferase reporter constructs reveal that IGF-I enhances BRCA1 promoter activity, suggesting that the effect of IGF-I is mediated at the transcriptional level. In addition, we provide evidence that the Sp1 zinc-finger protein is directly involved in BRCA1 gene transactivation. Combined, our data suggests that, at least part of the biological actions of IGF-I in mammary gland cells may be mediated through BRCA1. Dysregulated BRCA1 expression resulting from aberrant IGF signaling may have important consequences relevant to breast cancer pathogenesis.

ATM haplotypes and breast cancer risk in Jewish high-risk women.

While genetic factors clearly play a role in conferring breast cancer risk, the contribution of ATM gene mutations to breast cancer is still unsettled. To shed light on this issue, ATM haplotypes were constructed using eight SNPs spanning the ATM gene region (142 kb) in ethnically diverse non-Ashkenazi Jewish controls (n=118) and high-risk (n=142) women. Of the 28 haplotypes noted, four were encountered in frequencies of 5% or more and accounted for 85% of all haplotypes. Subsequently, ATM haplotyping of high-risk, non-Ashkenazi Jews was performed on 66 women with breast cancer and 76 asymptomatic. One SNP (rs228589) was significantly more prevalent among breast cancer cases compared with controls (P=4 x 10(-9)), and one discriminative ATM haplotype was significantly more prevalent among breast cancer cases (33.3%) compared with controls (3.8%), (P< or =10(-10)). There was no significant difference in the SNP and haplotype distribution between asymptomatic high-risk and symptomatic women as a function of disease status. We conclude that a specific ATM SNP and a specific haplotype are associated with increased breast cancer risk in high-risk non-Ashkenazi Jews.

The R72P P53 mutation is associated with familial breast cancer in Jewish women.

BRCA1/BRCA2 mutations account for a substantial proportion of familial breast cancer, but clearly mutations in additional genes exist, one candidate being the p53 gene. To evaluate its putative involvement in inherited predisposition to breast/ovarian cancer in Jewish high-risk women, mutational analysis of the p53 gene (exons 4-9) was carried out using exon-specific polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) analysis, complemented by DNA sequencing of abnormally migrating fragments. Overall, 132 Jewish breast cancer patient non-BRCA1/2 mutation carriers and 167 average risk controls (Ashkenazi (n=60), non-Ashkenazi (n=107)) were genotyped, and no inactivating p53 germline mutations were detected. Consistent migration abnormalities were noted in 167 fragments, 134 of which were shown to be the Arg72Pro polymorphism, whereas migration abnormalities in fragments containing exons 4 (n=2) and 6 (n=23) and introns 3 (n=4) and 9 (n=4) corresponded to five previously described polymorphisms. Allele distribution of the R72P missense mutation between ethnically diverse Jewish breast cancer cases and average risk controls showed significant differences: among non-Ashkenazi breast cancer cases, 62.5%, 33.3% and 4.2% were homozygous, heterozygous and homozygous for the Arg72, Arg72Pro and the Pro72 polymorphism, respectively, whereas for controls, the distribution was 22.4%, 65.4% and 12.2%, respectively (P=0.00052), and among Ashkenazi breast cancer cases, allele distribution was 68.5%, 29.6% and 1.9%, whereas for controls, the distribution was 50%, 40% and 10%, respectively (P=0.0125). We conclude that arginine homozygosity at codon 72 of the p53 gene is associated with a significant increased breast cancer risk in Jewish high-risk population.

Germline mutations in the PTEN gene in Israeli patients with Bannayan-Riley-Ruvalcaba syndrome and women with familial breast cancer.

Germline mutations in BRCA1 or BRCA2 account for the majority of inherited breast cancer cases. Yet, in up to 40% of familial breast cancer cases, no mutations can be detected in either gene. Germline mutations in PTEN underlie two inherited syndromes: Cowden disease (CD) and Bannayan-Riley-Ruvalcaba syndrome (BRRS). The known association of CD with breast cancer risk made it plausible that germline mutations within PTEN may play a role in inherited predisposition to breast cancer. The nine coding exons of the PTEN gene were screened for harboring germline mutations using denaturing gradient gel electrophoresis (DGGE) complemented by sequencing, in two subsets of Israeli patients: 12 patients clinically diagnosed with BRRS, and 89 women with an apparent inherited predisposition to breast cancer, some with salient features of CD. Two of three familial BRRS patients exhibited novel germline mutations in PTEN: a missense mutation changing methionine to arginine at codon 134, and insertion of two nucleotides (CA) at cDNA position 1215 resulting in a frameshift at codon 61 and a premature stop at codon 99. Among 89 high-risk women, two missense mutations were detected in exon 4: A to C change at cDNA position 1279 resulting in a change of aspargine to threonine at codon 82 (N82T), and a G to an A alteration in 1269 which alters threonine to alanine at codon 78 (T78A), a non-conservative missense mutation. This study suggests that PTEN does not play a major role in predisposing to hereditary breast cancer in Israeli women, and that detection of PTEN mutations in BRRS patients is more likely in familial cases.

Management of hemorrhoidal disease in patients with chronic spinal cord injury.

Hemorrhoidal disease is a common pathology in patients with chronic spinal cord injury (SCI). We describe our experience with the primary approach to this problem at the Proctology Division of the Sheba Medical Center. We treated 29 patients (26 men) with paraplegia due to SCI between 1995 and 1999. The mean age was 49 years (range, 22-74 years). All patients had hemorrhoids in stages ranging between II and IV. Main complaints were rectal bleeding (83%), difficulties in evacuation (38%) and discomfort or pain (28%). Eleven patients (38%) were treated conservatively (e. g. diet, hygiene and laxatives), while 18 patients (62%) underwent either banding or sclerotherapy of hemorrhoids or both. No major complication were observed. In 28 of 29 patients (96%), there was a significant reduction or cessation of bleeding and/or relief of symptoms; one patient (3%) required hemorrhoidectomy. Of the 28 successful treatments, 16 (57%) had partial reduction of bleeding or relief of symptoms, while in 12 (43%) response was complete. Of those who were treated conservatively, 9 (82%) had partial and 2 (18%) had complete relief of symptoms. Of those who had banding/sclerotherapy, 7 (41%) had partial and 10 (59%) had complete relief. We also examined the effect of perianal sensation on the treatment outcome. Of 16 patients with complete anesthesia, 11 (69%) had partial and 5 (31%) had complete relief, whereas of the 12 patients with preserved sensation, 5 (42%) had partial and 7 (58%) had complete relief. In conclusion, the approach of banding or sclerotherapy of hemorrhoids in SCI patients is safe and effective. When sensation of the perianal region is preserved, the outcome seems to be better. The cause of SCI has no impact on the treatment results. There was no difference in the outcome of treatment between patients with stage II and stage III hemorrhoids; patients with stage IV hemorrhoids seem to do worse than those with stages II and III.

Thromboangiitis obliterans: an old disease in need of a new look.

Almost a century after Buerger's original description of thromboangiitis obliterans, there is still no consensus about diagnostic criteria. The lack of a universally accepted method of diagnosis causes confusion, and mars research efforts. Some authors quote 'hematological disease' as one of the exclusion criteria. But in most recent reports, suspected Buerger patients did not undergo hematological tests to diagnose or rule out any primary or secondary hypercoagulable states. However, immunogenetic studies of Buerger's disease have led to a revived interest in the role of blood coagulation in the pathogenesis of thromboangiitis obliterans. Some association has been suggested between Buerger's disease and the antiphospholipid syndrome, as well as hyperhomocysteinemia. Other thrombophilic conditions have been described anecdotally in patients with Buerger's disease. In view of this developing line of investigation, there is a clear need to redefine the diagnostic algorithm and the criteria for diagnosing Buerger's disease.

Mutational analyses of BRCA1 and BRCA2 in Ashkenazi and non-Ashkenazi Jewish women with familial breast and ovarian cancer.

In Ashkenazi (East European) Jews, three predominant mutations in BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT) account for the majority of germline mutations in high-risk breast and/or ovarian cancer families. Among non-Ashkenazi Jews, the 185delAG, Tyr978Ter, and a handful of "private" mutations have been reported anecdotally within both genes. In this study we attempted to determine the spectrum of BRCA1 and BRCA2 mutations in high-risk Jewish individuals, non-carriers of any of the predominant Jewish mutations. We employed multiplex PCR and denaturing gradient gel electrophoresis (DGGE) analysis for BRCA2, and combined denaturing high performance liquid chromatography (DHPLC) and protein truncation test (PTT) for BRCA1, complemented by DNA sequencing. We screened 47 high-risk Jewish individuals, 26 Ashkenazis, and 21 non-Ashkenazis. Overall, 13 sequence alterations in BRCA1 and eight in BRCA2 were detected: nine neutral polymorphisms and 12 missense mutations, including five novel ones. The novel missense mutations did not co-segregate with disease in BRCA1 and were detected at rates of 6.25% to 52.5% in the general population for BRCA2. Our findings suggest that except for the predominant mutations in BRCA1 and BRCA2 in Jewish individuals, there are only a handful of pathogenic mutations within these genes. It may imply novel genes may underlie inherited susceptibility to breast/ovarian cancer in Jewish individuals.

Common origin of the I1307K APC polymorphism in Ashkenazi and non-Ashkenazi Jews.

A common germline missense mutation within the APC gene, I1307K, has recently been described in Ashkenazi Jews. We detected this polymorphism in two non-Ashkenazi Jewish women using denaturing gradient gel electrophoresis (DGGE), and hypothesized that in Jewish individuals it might not be restricted to Ashkenazim, and actually reflect a common ancestral polymorphism. To test this notion we performed allelic pattern determination using APC-linked markers in these two women and in nine Ashkenazi carrier controls. The pattern of the intragenic markers, as well as a single downstream marker 30-70 Kb from the APC gene was identical in all individuals, regardless of ethnic origin. We conclude that the I1307K polymorphism in Jewish individuals, is not restricted to Ashkenazim and probably reflects a founder mutation.

Positive margins of breast biopsy: is reexcision always necessary?

BACKGROUND AND OBJECTIVES: Breast-conserving surgery requires excision of all gross tumor and subsequent radiation therapy. It is generally accepted that the presence of microscopically positive margins requires reexcision. The goal of this study was to identify characteristics that distinguish breast biopsy specimens with positive margins that when reexcised are free from residual tumor. This population of patients may benefit from breast irradiation only, without the need for another surgical procedure. METHODS: One hundred and fifteen of 395 cancer-proven biopsies had positive surgical margins and were treated with reexcision or mastectomy. Sixty-seven of these were negative for residual tumor and 48 were positive for residual tumor. Evaluation for tumor type, tumor size, grade, presence of vascular invasion, volume of the biopsy specimen, true positivity and the number of positive margins, multifocality of the tumor, and type of anesthesia was done by univariate and multivariate analysis. RESULTS: Univariate and multivariate analysis revealed that factors associated with a positive reexcision included margin status, method of detection, histologic appearance, and type of anesthesia used. CONCLUSION: These results suggest that small, clinically detectable unifocal tumors could be treated without the need for a further excision. Eradication of microscopic residual tumor could be done by irradiation only, sparing the patient an additional surgical procedure.

Prophylactic mastectomy and inherited predisposition to breast carcinoma.

Relative to her risk of breast carcinoma, the woman with a BRCA1 or BRCA2 gene mutation can be managed either by intensive screening (with or without chemoprevention) or by prophylactic mastectomy. Although it would be preferable to avoid prophylactic surgery, the current level of screening technology and the rudimentary state of chemoprevention do not guarantee a good outcome with intensive surveillance. A review of the currently available data was undertaken to determine the efficacy of prophylactic surgery, intensive screening, and chemoprevention. An attempt then was made to extrapolate the efficacy of the various approaches to the management of women who carry BRCA1 or BRCA2 gene mutations. Intensive surveillance may not detect breast carcinoma at an early, curable stage in young women with BRCA1 or BRCA2 gene mutations because the growth rate of the tumors in these women most likely will be rapid and the density of the breast tissue may compromise detection. Chemoprevention is in its infancy, and its efficacy in this population is unknown. Conversely, prophylactic surgery may not be completely effective in preventing breast carcinoma. The authors are hopeful that sometime in the next decade advances in chemoprevention, screening technology, or breast carcinoma treatment will make mastectomy obsolete. However, for the time being prophylactic mastectomy has attributes that make it an alternative for this population that must be considered. Careful discussion of all options is essential in the management of these women.

Spontaneous leak and transection of permanent subclavian catheters.

BACKGROUND AND OBJECTIVES: Implanted central venous access ports are frequently used. Spontaneous break and catheter transection are serious but rare complications of permanent subclavian catheters. We report our experience with this serious complication and identify possible warning signs. MATERIALS AND METHODS: Between 1990 and 1996, 285 permanent subclavian catheters were placed at the Sheba Medical Center, Tel Hashomer, Israel. RESULTS: We evaluated the patient population for this complication and searched for possible warning signs. A total of 12 patients (4.2%) with this complication were identified, 8 with transection and distal embolization and 4 with a partial tear only. The pinch-off sign was noted as an early warning in only 5 patients. All other patients developed symptoms only immediately before the diagnosis of this complication. The mean duration from insertion to identification of tear or transection was 9.6 months (range 1-24 months). CONCLUSION: Awareness and clinical suspicion are most important in identification and prevention of this serious complication. Catheters should be taken out when treatment is completed or after 12 months in order to prevent catheter breaks.

An identical novel mutation in BRCA1 and a common haplotype in familial ovarian cancer in non-Ashkenazi Jews.

Unique germline mutations in BRCA1 and BRCA2 account for inherited predisposition to breast and ovarian cancer in high-risk families. In Jewish high-risk individuals of Ashkenazi (east European) descent, three predominant mutations, 185delAG and 5382insC (BRCA1) and 6174delT (BRCA2), seem to account for a substantial portion of germline mutations, and two of these mutations (185delAG and 6174delT) are also found at about 1% each in the general Jewish-Ashkenazi population. We identified a novel BRCA1 mutation in two Jewish-non-Ashkenazi families with ovarian cancer: a thymidine to guanidine alteration at position 3053, resulting in substitution of tyrosine at codon 1017 for a stop codon (Tyr1017Ter). The mutation was first detected by protein truncation test (PTT) and confirmed by sequencing and a modified restriction digest assay. Allelotyping of mutation carriers using intragenic BRCA1 markers revealed that the haplotype was identical in these seemingly unrelated families. No mutation carrier was found among 118 unselected Jewish individuals of Iranian origin. Our findings suggest that this novel mutation should be incorporated into the panel of mutations analysed in high-risk families of the appropriate ethnic background, and that the repertoire of BRCA1 mutations in Jewish high-risk families may be limited, regardless of ethnic origin.

TNF-induced modulations of phospholipid metabolism in human breast cancer cells.

Tumor necrosis factor alpha (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G0+G1/S, accompanied by apoptosis. 31P and 13C NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and 2H NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52+/-6%. Later (approximately 15 h), the activity of phosphocholine:cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis.

Sentinel lymph node involvement--a predictor for axillary node status with breast cancer--has the time come?

AIMS: Axillary node dissection for breast cancer is important for staging and its prognostic value. Sentinel nodes are defined as the first nodes into which the primary cancer drains. This study investigates whether identification, removal and pathological examination of these nodes indicates whether the completion of axillary lymphadenectomy is required. METHODS: Using a vital dye injected at the primary tumour site, we were able to identify sentinel nodes in 96 out of 98 women examined. RESULTS: An average number of 2.7 +/- 1.2 nodes per patient were identified as sentinel nodes. In 83% of cases there was a correlation between the involvement of the sentinel nodes and the rest of the axillary nodes. In 14% of patients the sentinel nodes were the only nodes involved with tumour. In three cases the sentinel nodes were negative, but other axillary nodes were tumour-positive. CONCLUSION: The major problem in routine application of this method to the decision to perform axillary lymph node dissection (ALND) is the time needed for pathological identification of lymph node involvement by tumor.

The 185delAG BRCA1 mutation originated before the dispersion of Jews in the diaspora and is not limited to Ashkenazim.

The 185delAG mutation in BRCA1 is detected in Ashkenazi Jews both in familial breast and ovarian cancer and in the general population. All tested Ashkenazi mutation carriers share the same allelic pattern at the BRCA1 locus. Our previous study showed that this 'Ashkenazi' mutation also occurs in Iraqi Jews with a similar allelic pattern. We extended our analysis to other non-Ashkenazi subsets: 354 of Moroccan origin, 200 Yemenites and 150 Iranian Jews. Heteroduplex analysis complemented by direct DNA sequencing of abnormally migrating bands were employed. Four of Moroccan origin (1. 1%) and none of the Yemenites or Iranians was a carrier of the 185delAG mutation. BRCA1 allelic patterns were determined for four of these individuals and for 12 additional non-Ashkenazi 185delAG mutation carriers who had breast/ovarian cancer. Six non-Ashkenazi individuals shared the common 'Ashkenazi haplotype', four had a closely related pattern, and the rest ( n = 6) displayed a distinct BRCA1 allelic pattern. We conclude that the 185delAG BRCA1 mutation occurs in some non-Ashkenazi populations at rates comparable with that of Ashkenazim. The majority of Jewish 185delAG mutation carriers have a common allelic pattern, supporting the founder effect notion, but dating the mutation's origin to an earlier date than currently estimated. However, the different allelic pattern at the BRCA1 locus even in some Jewish mutation carriers, might suggest that the mutation arose independently.

[Experience at Sheba Hospital in oncogenetic counseling and genetic testing of women with a high risk for breast and ovarian cancer].

There is inherited predisposition to breast and ovarian cancer in 5-10% of all women with these diseases. Germline mutations in BRCA1 and BRCA2 presumably account for most of the genetically susceptible individuals. We summarize 2 years of experience in counseling and testing for inherited predisposition to these cancers. 597 women (from 320 families) have been evaluated since August 1995. 242 were evaluated for inherited predisposition to breast and ovarian cancer. One-third had clear-cut evidence of familial background. 74 families were of Ashkenazi origin; the age range of breast cancer was 30-35, of ovarian cancer 40-45. In 80% of families other cancers were also noted in first degree family members, including lung, colon, and prostate cancer and leukemia. Genetic testing revealed that 45% of affected and 25% of unaffected women were carriers of a mutation in BRCA1 or BRCA2: 67/90 185delAG (BRCA1), 12/90 6174delT (BRCA2), and 4/90 of 5382insC (BRCA1). In addition, a novel mutation in exon 11 of BRCA1 was detected, carried by 7/90 women. The experience gained in oncogenetic counseling and genetic testing for inherited cancer predisposition will eventually enable determining an optimal, rational therapeutic regimen in carriers of mutations.

Avoiding diarrhea after subtotal colectomy with primary anastomosis in the treatment of colon cancer.

BACKGROUND: Subtotal colectomy has been criticized as causing increased frequency of stool passage, thus adding to the patients' morbidity. We review our experience with subtotal colectomy and determine the factors affecting postoperative diarrhea. STUDY DESIGN: One hundred thirty-six patients with colon cancer were treated by primary subtotal colectomy. Of these, 30 percent underwent an emergency resection; 15 percent, semi-emergency resection; and 55 percent, elective subtotal colectomy. There were 29 complications and 3 deaths. RESULTS: The incidence of complications was higher when the operation was carried out on an emergency or semi-emergency basis. In assessing the patients' increased postoperative stool frequency, there was no difference between the groups; but, the length of the remaining colon and the resected terminal ileum had a significant effect on postoperative diarrhea. If less than 10 cm of terminal ileum is resected and more than 10 cm of colon is left above the peritoneal reflection, there is a marked decrease in the incidence of diarrhea after subtotal colectomy. CONCLUSIONS: Subtotal colectomy is an acceptable treatment for left colonic carcinoma, electively as well as in emergency situations. Postoperative diarrhea can be minimized by attention to the length of small bowel and sigmoid that are resected.