Combination of recombinant follicle stimulating hormone with human menopausal gonadotrophin or recombinant luteinizing hormone in a long gonadotrophin-releasing hormone agonist protocol: a retrospective study.

PURPOSE: To assess the effect of supplementation with recombinant human luteinizing hormone (rhLH) for patients treated either with recombinant follicle stimulating hormone (rFSH) plus rhLH or with rFSH plus human menopausal gonadotrophin (HMG) in a long gonadotrophin-releasing hormone (GnRH) agonist-stimulation protocol. METHODS: A single-centre, retrospective analysis of patients with hypo responsiveness to a long GnRH agonist protocol (n = 174), with consecutive in-vitro fertilization or intracytoplasmic sperm injection cycles, compared the outcomes of long luteal GnRH agonist ovarian stimulation using rFSH combined with HMG (n = 100) versus rFSH combined with rhLH (n = 74). The endpoints included clinical pregnancy, number of oocytes retrieved, and total gonadotrophin dose. RESULTS: Significantly more clinical pregnancies were achieved after stimulation with rFSH and rhLH than after stimulation with rFSH and HMG (35.1 vs. 19%, p < 0.01). More oocytes were recovered (13.1 vs. 11.3, p = 0.024) with less FSH utilized in the rFSH and rhLH group than in the rFSH and HMG group (2706.4 vs. 4134.2 U, p < 0.001). CONCLUSIONS: Use of rFSH combined with rhLH in long GnRH agonist assisted reproductive technology (ART) cycles was associated with more clinical pregnancies, recovery of more oocytes, and reduction in gonadotrophin use, suggesting that the superior purity and consistency of rFSH and rhLH may result in better clinical outcomes.

Overexpression of NBS1 induces epithelial-mesenchymal transition and co-expression of NBS1 and Snail predicts metastasis of head and neck cancer.

Major causes of head and neck squamous cell carcinoma (HNSCC)-related deaths are cervical node and distant metastasis. We previously demonstrated that overexpression of the DNA double-strand break repair protein Nijmegen breakage syndrome 1 (NBS1) is a prognostic marker of advanced HNSCCs. Epithelial-mesenchymal transition (EMT) was demonstrated to be the major mechanism responsible for mediating invasiveness and metastasis of late-stage cancers. We therefore investigated the role of NBS1 overexpression in mediating EMT and metastasis. NBS1 overexpression was associated with metastasis of HNSCC patients using tissue microarray-immunohistochemistry approach. Induction of EMT was observed in an NBS1-overexpressing HNSCC cell line (FADUNBS), whereas short-interference RNA (siRNA)-mediated repression of endogenous NBS1 reversed the shift of EMT markers. Increased migration/invasiveness of FADUNBS was shown by in vitro and in vivo assays. NBS1 overexpression upregulated the expression of an EMT regulator Snail and its downstream target matrix metalloproteinase-2. EMT phenotypes and increased migration/invasiveness of FADUNBS cells were reversed by siRNA-mediated repression of Snail expression or a phosphatidylinositol 3-kinase-specific inhibitor. In HNSCC samples, co-expression of NBS1/Snail in primary tumors correlated with metastasis and the worst prognosis. These results indicate that NBS1 overexpression induces EMT through the upregulation of Snail expression, and co-expression of NBS1/Snail predicts metastasis in HNSCCs.

An argument against routine sentinel node mapping for DCIS.

Indications for sentinel lymph node mapping (SLNM) for patients with ductal carcinoma in situ (DCIS) of the breast are controversial. We reviewed our institutional experience with SLNM for DCIS to determine the node positive rate and clarify indications for nodal staging in patients with DCIS. Since 1998 we have used SLNM to stage breast cancer patients using both blue dye and radiocolloid. In DCIS patients, SLNM has been reserved for patients considered at high risk for harboring coexistent invasive carcinoma or treated by mastectomy. All sentinel nodes were evaluated with serial sectioning, hematoxylin and eosin staining, and immunohistochemical evaluation for cytokeratins. We identified 44 patients with 46 cases of DCIS (two patients with bilateral disease). SLNM identified at least one sentinel node in all cases. In all cases, the sentinel node(s) were negative for axillary metastasis. We calculated the binomial probability of observing 0 of 46 cases as negative when the expected incidence according to published reports in the surgical literature was 13 per cent and found a P value of <0.01. Based on this case-series observation, we conclude SLNM should not be routinely performed for patients with DCIS. We now use SLNM only for DCIS patients treated by mastectomy.

Detection of esophageal carcinoma using single-photon emission computed tomography of thallium-201: a preliminary report.

The aim of this preliminary study was to evaluate the potential usefulness of thallium-201 (Tl-201) in detecting esophageal carcinoma. A total of 15 patients with esophageal carcinoma were arranged for Tl-201 single-photon emission computed tomography (SPECT) of the chest. Thirteen of the patients had epidermoid carcinoma and two had adenocarcinoma. Meanwhile, 7 normal controls without any history of esophageal disease also accepted Tl-201 SPECT of the chest for comparison. From the 15 patients, Tl-201 chest SPECT detected esophageal carcinoma in 13 (86.7%) but not in 2 (13.3%) patients with epidermoid carcinoma. In contrast, all 7 normal controls (100.0%) had negative results of Tl-201 chest SPECT. Our study showed that the Tl-201 chest SPECT scan could be considered as a non-invasive useful imaging method in detection of esophageal carcinoma.

Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process.

Type II survivors arise in Saccharomyces cells lacking telomerase by a recombinational pathway that results in very long and heterogeneous length telomeres. Here we show that type II telomeres appeared abruptly in a population of cells with very short telomeres. Once established, these long telomeres progressively shortened. Short telomeres were substrates for rare, one-step lengthening events. The generation of type II survivors was absolutely Rad50p dependent. In a telomerase-proficient cell, the telomere-binding Rif proteins inhibited telomerase lengthening of telomeres. In a telomerase-deficient strain, Rif proteins, especially Rif2p, inhibited type II recombination. These data argue that only short telomeres are substrates for type II recombination and suggest that the donor for this recombination is not a chromosomal telomere.

Pif1p helicase, a catalytic inhibitor of telomerase in yeast.

Mutations in the yeast Saccharomyces cerevisiae PIF1 gene, which encodes a 5'-to-3' DNA helicase, cause telomere lengthening and a large increase in the formation rate of new telomeres. Here, we show that Pif1p acts by inhibiting telomerase rather than telomere-telomere recombination, and this inhibition requires the helicase activity of Pif1p. Overexpression of enzymatically active Pif1p causes telomere shortening. Thus, Pif1p is a catalytic inhibitor of telomerase-mediated telomere lengthening. Because Pif1p is associated with telomeric DNA in vivo, its effects on telomeres are likely direct. Pif1p-like helicases are found in diverse organisms, including humans. We propose that Pif1p-mediated inhibition of telomerase promotes genetic stability by suppressing telomerase-mediated healing of double-strand breaks.

Telomere-telomere recombination is an efficient bypass pathway for telomere maintenance in Saccharomyces cerevisiae.

Many Saccharomyces telomeres bear one or more copies of the repetitive Y' element followed by approximately 350 bp of telomerase-generated C(1-3)A/TG(1-3) repeats. Although most cells lacking a gene required for the telomerase pathway die after 50 to 100 cell divisions, survivors arise spontaneously in such cultures. These survivors have one of two distinct patterns of telomeric DNA (V. Lundblad and E. H. Blackburn, Cell 73:347-360, 1993). The more common of the two patterns, seen in type I survivors, is tandem amplification of Y' followed by very short tracts of C(1-3)A/TG(1-3) DNA. By determining the structure of singly tagged telomeres, chromosomes in type II survivors were shown to end in very long and heterogeneous-length tracts of C(1-3)A/TG(1-3) DNA, with some telomeres having 12 kb or more of C(1-3)A/TG(1-3) repeats. Maintenance of these long telomeres required the continuous presence of Rad52p. Whereas type I survivors often converted to the type II structure of telomeric DNA, the type II pattern was maintained for at least 250 cell divisions. However, during outgrowth, the structure of type II telomeres was dynamic, displaying gradual shortening as well as other structural changes that could be explained by continuous gene conversion events with other telomeres. Although most type II survivors had a growth rate similar to that of telomerase-proficient cells, their telomeres slowly returned to wild-type lengths when telomerase was reintroduced. The very long and heterogeneous-length telomeres characteristic of type II survivors in Saccharomyces are reminiscent of the telomeres in immortal human cell lines and tumors that maintain telomeric DNA in the absence of telomerase.

Retrotransposon reverse-transcriptase-mediated repair of chromosomal breaks.

The abundance of short and long interspersed nuclear sequences (SINEs and LINEs) and pseudogenes in eukaryotic genomes indicates that reverse transcriptase (RT)-mediated phenomena are important in genome evolution. However, the mechanisms involved in their spread are largely unknown. We have developed a selection system in the yeast Saccharomyces cerevisiae to test whether RT-mediated events could be linked to the repair of double-strand breaks (DSBs). Here we show that DSBs can be fixed by the insertion of complementary DNAs at the break site. In the presence of functional RT (from human L1, yeast Tyl or Crithidia CRE1), and in the absence of homologous recombination, an HO endonuclease-induced DSB at the mating type (MAT) locus is the primary site at which a marked cDNA is observed among surviving cells. The structure and junctional sequences of these insertions suggest that repair occurs primarily by non-homologous recombination. Our data support a role for endogenous retroelements in the repair of chromosomal breaks.

A new non-LTR retrotransposon provides evidence for multiple distinct site-specific elements in Crithidia fasciculata miniexon arrays.

We have identified a new member of the family of trypanosome site-specific retrotransposons, using a degenerate oligonucleotide PCR strategy. The 9595 bp element, termed Crithidia retrotransposable element 2 (CRE2), was cloned and found to be inserted in the tandemly arrayed miniexon genes of Crithidia fasciculata. The element is flanked by 29 bp target site duplications but lacks the 3' poly dA tract characteristic of most other non-long terminal repeat retrotransposons. The amino terminal region of the single 2518-codon open reading frame contains a putative metal-binding motif and a proline-rich region similar to gag-like domains of other retrotransposons. The carboxy terminal region of this open reading frame shares sequence homology with the reverse transcriptase and putative endonuclease regions of three previously described trypanosomatid site-specific retrotransposons. All four of these retrotransposons are specifically inserted between nucleotides 11 and 12 of the highly conserved 39mer sequence of the miniexon gene. Most copies of CRE2 and the previously characterized CRE1 are located on different sized chromosomes. Additional CRE-related sequences were identified by screening Crithidia libraries. These results suggest that a particular sequence in the C. fasciculata miniexon repeat is the target for multiple distinct site-specific retrotransposon insertions.