The Drosophila ovary is an attractive system to study how niches control stem cell self-renewal and differentiation. The niche for germline stem cells (GSCs) provides a Dpp/Bmp signal, which is essential for GSC maintenance.

Stem cells are responsible for replacing damaged or dying cells in various adult tissues throughout a lifetime. They possess great potential for future regenerative medicine and gene therapy. However, the mechanisms governing stem cell regulation are poorly understood. Germline stem cells (GSCs) in the Drosophila testis have been shown to reside in niches, and thus these represent an excellent system for studying relationships between niches and stem cells. Here we show that Bmp signals from somatic cells are essential for maintaining GSCs in the Drosophila testis. Somatic cyst cells and hub cells express two Bmp molecules, Gbb and Dpp. Our genetic analysis indicates that gbb functions cooperatively with dpp to maintain male GSCs, although gbb alone is essential for GSC maintenance. Furthermore, mutant clonal analysis shows that Bmp signals directly act on GSCs and control their maintenance. In GSCs defective in Bmp signaling,expression of bam is upregulated, whereas forced bamexpression in GSCs causes the GSCs to be lost. This study demonstrates that Bmp signals from the somatic cells maintain GSCs, at least in part, by repressing bam expression in the Drosophila testis. dpp signaling is known to be essential for maintaining GSCs in the Drosophila ovary. This study further suggests that both Drosophila male and female GSCs use Bmp signals to maintain GSCs.

The relationship between loss of functional p53 and human reduced folate carrier (hRFC) levels and function was examined in REH lymphoblastic leukemia cells, which express wild type p53, and in p53-null K562 cells (K562 pTet-on/p53 ) engineered to express wild type p53 under control of a tetracycline-inducible promoter. Activation of p53 in REH cells by treatment with daunorubicin was accompanied by decreased (ϳ5-fold) levels of hRFC transcripts and methotrexate transport. Treatment of K562 pTet-on/p53 cells with doxycycline resulted in a dosedependent expression of p53 protein and transcripts, increased p21 protein, decreased dihydrofolate reductase, and G 1 arrest with decreased numbers of cells in S-phase. p53 induction was accompanied by up to 3-fold decreases in hRFC transcripts transcribed from the upstream hRFC-B promoter and similar losses of hRFC protein and methotrexate uptake capacity. Expression of p15 in an analogous inducible system in K562 cells resulted in a nearly identical decrease of S-phase cells and dihydrofolate reductase without effects on hRFC levels or activity. When the hRFC-B promoter was expressed as full-length and basal promoter-luciferase reporter constructs in K562 pTet-on/p53 cells, induction of p53 with doxycycline resulted in a 3-fold loss of promoter activity, which was reversed by cotransfection with a trans-dominant-negative p53. These studies show that wild type p53 acts as a repressor of hRFC gene expression, via a mechanism that is independent of its effects on cell cycle progression.

Summary. The downstream effects of p15 and p16 gene deletions and loss of transcripts on dihydrofolate reductase (DHFR) were examined in 63 B-precursor (BP) acute lymphoblastic leukaemia (ALL) samples. p15 and/or p16 gene deletions were seen in 6% and 8%, respectively, of BP-ALL samples; however, losses of p15 and/or p16 transcripts were seen in 26 out of 63 (41%) samples. Loss of p15 transcripts (36´5%) exceeded that for p16 (17´5%). For the 26 BP-ALLs that lacked p15 and/or p16 transcripts, only six (23%) exhibited low levels of DHFR by flow cytometry assay with Pt430, a fluorescent anti-folate. Conversely, 18 out of 37 (49%) BP-ALL samples with intact p15 and/or p16 genes and transcripts showed low levels of DHFR (P 0´04). In p15-and p16-null K562 cells transfected with a tetracycline-inducible p15 cDNA construct, induction of p15 transcripts and protein was accompanied by decreased growth rates, decreased S-phase fraction, decreased retinoblastoma protein phosphorylation, and markedly reduced levels of DHFR transcripts and protein.Collectively, our results suggest that losses of p15 and/or p16 gene expression result in elevated levels of DHFR in BP-ALL in children. However, additional downstream factors undoubtedly also contribute to elevated levels of this enzyme target.

PRT-201 is a recombinant human pancreatic elastase under development as a treatment for blood vessels to promote hemodialysis access patency. Proteases such as elastase are normally inactivated by antiproteases such as alpha 1-antitrypsin. It is unknown if serum from patients with alpha 1-antitrypsin deficiency will inhibit PRT-201 elastase activity. An assay for PRT-201 elastase activity in the presence of serum was developed and validated. PRT-201 elastase activity inhibition curves were developed using serum and also using purified alpha 1-antitrypsin and alpha 2-macroglobulin. Serum from 15 patients with documented alpha 1-antitrypsin deficiency, some of whom were receiving alpha 1-antitrypsin augmentation therapy, and four normal volunteers was analyzed. Serum from normal volunteers and patients with alpha 1-antitrypsin deficiency completely inactivated PRT-201 elastase activity in vitro. In the alpha 1-antitrypsin-deficient patients, the volume of serum necessary to inhibit elastase activity was related to the serum concentration of alpha 1-antitrypsin and augmentation therapy. Purified alpha 1-antitrypsin and alpha 2-macroglobulin were each alone capable of completely inhibiting PRT-201 elastase activity. It is unlikely that the use of PRT-201 will be associated with negative outcomes in patients with alpha 1-antitrypsin deficiency.