Dysregulated STAT1-SOCS1 control of JAK2 promotes mammary luminal progenitor cell survival and drives ERα(+) tumorigenesis.

We previously reported that STAT1 expression is frequently abrogated in human estrogen receptor-α-positive (ERα(+)) breast cancers and mice lacking STAT1 spontaneously develop ERα(+) mammary tumors. However, the precise mechanism by which STAT1 suppresses mammary gland tumorigenesis has not been fully elucidated. Here we show that STAT1-deficient mammary epithelial cells (MECs) display persistent prolactin receptor (PrlR) signaling, resulting in activation of JAK2, STAT3 and STAT5A/5B, expansion of CD61(+) luminal progenitor cells and development of ERα(+) mammary tumors. A failure to upregulate SOCS1, a STAT1-induced inhibitor of JAK2, leads to unopposed oncogenic PrlR signaling in STAT1(-/-) MECs. Prophylactic use of a pharmacological JAK2 inhibitor restrains the proportion of luminal progenitors and prevents disease induction. Systemic inhibition of activated JAK2 induces tumor cell death and produces therapeutic regression of pre-existing endocrine-sensitive and refractory mammary tumors. Thus, STAT1 suppresses tumor formation in mammary glands by preventing the natural developmental function of a growth factor signaling pathway from becoming pro-oncogenic. In addition, targeted inhibition of JAK2 may have significant therapeutic potential in controlling ERα(+) breast cancer in humans.

Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection.

This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.

A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes.

OBJECTIVE: To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. DESIGN: The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. SETTING: Clinical and academic research environment. PATIENT(S): 59 patients undergoing fertility treatment. INTERVENTION(S): Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. MAIN OUTCOME MEASURE(S): Sperm head DNA density and sperm variables. RESULT(S): A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. CONCLUSION(S): The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

Characterization of human herpesvirus 8 ORF59 protein (PF-8) and mapping of the processivity and viral DNA polymerase-interacting domains.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118-126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.

Human herpesvirus-8 ORF K8.1 gene encodes immunogenic glycoproteins generated by spliced transcripts.

A cDNA library from phorbol ester-induced human herpesvirus-8 (HHV-8) carrying BCBL-1 cells was screened with an HIV+KS+ serum, and several cDNA clones encoding HHV-8 proteins were identified. Sequence analysis of two full-length cDNA clones show open reading frames (ORFs) encoded by spliced messages originating from the HHV-8 K8.1 gene. One cDNA encodes an ORF of 228 amino acids, designated K8. 1.A, with a cleavable signal sequence, a transmembrane domain, and four N-glycosylation sites. The splicing event generated the transmembrane domain in the ORF not seen in the genomic K8.1 ORF. Another cDNA encodes an ORF of 167 amino acids, designated K8.1.B, that shares similar amino and carboxyl termini with ORF K8.1.A but with an in-frame deletion. The primary translation products of ORF K8.1A (34 kDa) and K8.1B (20 kDa) in the in vitro-transcription-translation experiments shifted into glycosylated species of 43 and 32 kDa, respectively, in the presence of microsomal membranes. This suggested that the ORF K8.1A and K8.1B encode for glycoproteins. Riboprobes from the K8.1A cDNA insert hybridized with an HHV-8-specific 0.9-kb abundant transcript from BCBL-1 cells. Synthesis of this RNA was eliminated in the presence of a DNA synthesis inhibitor, suggesting that this RNA was a late gene transcript. Because ORFs K8.1A and K8.1B are unique for HHV-8, human sera were tested in Western blot reactions for antibodies against glutathione-S-transferase-ORF K8.1A fusion protein. All sera that were positive for HHV-8 antibodies in immunofluorescence assays with phorbol ester-induced BCBL-1 cells were also positive for anti-ORF K8.1A antibodies. This suggests that measurement of anti-ORF K8.1A antibodies would provide an HHV-8-specific serological assay. Further work is needed to define the biological role of the HHV-8 ORF K8.1A and K8.1B glycoproteins.

Identification and characterization of human herpesvirus-8 lytic cycle-associated ORF 59 protein and the encoding cDNA by monoclonal antibody.

Monoclonal antibody (Mab) 11D1 specific for HHV-8 showed a predominantly nuclear membrane fluorescence with about 30% of phorbol ester (TPA)-induced HHV-8-carrying BCBL-1 cells and with 2-8% of uninduced cells, but not with other herpes viruses infected cells. This Mab immunoprecipitated a 50-kDa polypeptide from BCBL-1 cells. The synthesis of this polypeptide was reduced but not inhibited by phosphonoacetic acid (PAA). A 2.3-kb cDNA insert from a cDNA library of TPA-induced BCBL-1 cells was identified by Mab 11D1. Sequence analysis shows that this cDNA is open at the 5' end and encodes two ORFs of 396AA (5' end) and 357AA (3' end). These ORFs are identical to the published HHV-8 ORFs 59 and 58, respectively in vitro transcription and translation of the cDNA resulted in the synthesis of a 50-kDa polypeptide and its partial peptide map was identical to that of the 50-kDa polypeptide detected in the TPA induced BCBL-1 cells. Riboprobe made from the cDNA insert hybridized with several viral specific RNAs from BCBL-1 cells. Levels of these RNA species were reduced, but not inhibited by PAA. These characteristics are similar to other herpes viruses genes encoding the lytic cycle associated early-late class accessory proteins that are essential for viral DNA replication. This Mab 11D1 recognizing the HHV-8 lytic cycle associated ORF 59 protein will be highly useful in monitoring the lytic replicative cycle.