Characterization of PTEN mutations in brain cancer reveals that pten mono-ubiquitination promotes protein stability and nuclear localization.

PTEN is a PIP3 phosphatase that antagonizes oncogenic PI3-kinase signalling. Due to its critical role in suppressing the potent signalling pathway, it is one of the most mutated tumour suppressors, especially in brain tumours. It is generally thought that PTEN deficiencies predominantly result from either loss of expression or enzymatic activity. By analysing PTEN in malignant glioblastoma primary cells derived from 16 of our patients, we report mutations that block localization of PTEN at the plasma membrane and nucleus without affecting lipid phosphatase activity. Cellular and biochemical analyses as well as structural modelling revealed that two mutations disrupt intramolecular interaction of PTEN and open its conformation, enhancing polyubiquitination of PTEN and decreasing protein stability. Moreover, promoting mono-ubiquitination increases protein stability and nuclear localization of mutant PTEN. Thus, our findings provide a molecular mechanism for cancer-associated PTEN defects and may lead to a brain cancer treatment that targets PTEN mono-ubiquitination.

A new class of cancer-associated PTEN mutations defined by membrane translocation defects.

Phosphatase and tensin homolog (PTEN), which negatively regulates tumorigenic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signaling, is a commonly mutated tumor suppressor. The majority of cancer-associated PTEN mutations block its essential PIP3 phosphatase activity. However, there is a group of clinically identified PTEN mutations that maintain enzymatic activity, and it is unknown how these mutations contribute to tumor pathogenesis. Here, we show that these enzymatically competent PTEN mutants fail to translocate to the plasma membrane where PTEN converts PIP3 to PI(4,5)P2. Artificial membrane tethering of the PTEN mutants effectively restores tumor suppressor activity and represses excess PIP3 signaling in cells. Thus, our findings reveal a novel mechanism of tumorigenic PTEN deficiency.

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts.

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.

New reference levels for CA125 in pre- and postmenopausal women.

Objective: CA125 is a glycoprotein commonly produced by the endometrium. Serum CA125 is widely useful as a marker for ovarian cancer screening, with 35 U/mL the generally accepted upper limit of normal. Since CA125 is known to fluctuate with vaginal bleeding and menopausal status, the purpose of this study was to develop different reference ranges for normal women of differing physiologic states.Methods: We retrospectively analyzed 1,407 patients with serum CA125 levels taken at Cleveland Clinic Florida between 1992 and 1997. Patients with obvious elevation of CA125 from gynecologic cancers were excluded. Student t test was used to determine statistical significance.Results: Among 1,407 patients studied, 256 were premenopausal, 46 perimenopausal, 957 postmenopausal, and 148 excluded due to known gynecologic cancers. There was a significant difference between the mean CA125 values of premenopausal (19.3 +/- 15.6) and postmenopausal women (11.7 +/- 9.2) with P.7 x 10(-6). Among premenopausal women, there was a statistically significant difference between CA125 levels during menses (21.4 +/- 19.3) and luteal phase (14.0 +/- 9.1) with P =.03. Among the subgroup of postmenopausal women with known bleeding history, the mean CA125 values for women with vaginal bleeding was 12.49 +/- 11.5 compared to those without bleeding, 9.62 +/- 4.6 (P =.017).Conclusions: For normal premenopausal women, the overall upper limit of CA125 should be 50 U/mL. However, if menstrual status is known, the upper normals should be: 62 during menses, 51 for proliferative phase, and 32 for luteal phase. For postmenopausal women, the CA125 levels should be no more than 35 for those with vaginal bleeding and 20 for those without bleeding. The stratified CA125 values may help provide more specific ovarian cancer screening.