Gem Cell Research Articles

Significance of acid sphingomyelinase and ceramide in inducing gemcitabine-resistant pancreatic cancer cell line PANC-1

Objective To establish gemcitabine-resistant pancreatic cancer cell line PANC-1/ gem and discuss its biological characters and drug resistance mechanism. Methods gemcitabine-resist-ant pancreatic cancer cell line was obtained by culture of pancreatic cancer cell line PANC-1 in vitro with intermittently increasing the concentration of gemcitabine in the culture medium for 24 weeks. After that, TUNEL technique was employed to detect the apoptosis. Meanwhile, its drug sensitivity as well as the expression of sphingomyelinase, contents of C2-ceramide were determined respectively. Results Drug-resistant PANC-1/Gem cell was successfully induced with gemeitabine for 24 weeks. The IC_(50) increased from (8.13±0.85)μg/ml in PANC-1 to (285.40±34.83)μg/ml in PANC-1/Gem. Compared with the normal pancreatic cancer cell line PANC-1, the drug resistance indexes of PANC-1/gem to gemcitabine was 35.1 and the apoptosis rate was reduced from 6.21 to 2.74%. The ex-pression of sphingomyelinase was lower than that of pancreatic cancer cell line PANC-1, and the con-tents of ceramide was reduced from (364.95±46.11 pmol/mg protein) to (120.61±20.07 pmol/mg protein). Conclusion The durg resistance of gemcitabine to pancreatic cancer cell line is positively rel-evant to the low expression of acid sphingomyelinase as well as ceramide deficiency. Key words: Pancreatic neoplasms; Gemcitabine; Drug resistance; Acid sphingomyeli-nase; Ceramide

Meiotic behavior of aneuploid chromatin in mouse models of Down syndrome

Aneuploidy, which leads to unpaired chromosomal axes during meiosis, is frequently accompanied by infertility. We previously showed, using three mouse models of Down syndrome, that it is an extra chromosome, but not extra gene dose, that is associated with male infertility and virtual absence of post-meiotic gem cells. Here, we test the hypothesis that aneuploid segments are differentially modified and expressed during meiosis, depending on whether they are present as an extra chromosome or not. In all three models examined, the trisomic region lacks a pairing partner, but in one case, spermatocytes have an extra (and unpaired) chromosome, while the two other models involve translocation of the trisomic region rather than an extra chromosome. An extra unpaired chromosome was always modified by phosphorylation of histone H2AX and lacked RNA PolII. But in the case of trisomic regions attached to a paired chromosome, assembly of these protein modifications was affected by the position of a trisomic region relative to a centromere and the physical extent of the unpaired chromatin. Analysis of gene expression in testes revealed that extra copy number alone was not sufficient for meiotic upregulation of genes in the trisomic interval. Additionally and unexpectedly, presence of meiotic gene silencing chromatin modifications was not sufficient for downregulation of genes in unpaired trisomic chromatin. Thus, the meiotic chromatin modifications that are cytologically visible are unlikely to be directly involved in sterility versus fertility of DS models. Finally, the presence of an extra unpaired chromosome, but not the presence of extra (trisomic) genes, caused global deregulation of transcription in spermatocytes. These results reveal mechanisms by which an extra chromosome, but not trisomic gene dose, impact on meiotic progress and infertility.

Radiotherapy versus carboplatin for stage I seminoma: Updated analysis of the MRC/EORTC randomized trial (ISRCTN27163214)

1 Background: At ASCO 2004 we reported a randomised trial (MRC TE19, EORTC 30982) comparing 1 course carboplatin (C) at AUC×7 with adjuvant radiotherapy (RT) for stage I seminoma. With median follow-up (FU) of 4 yrs, C was shown to be non-inferior for relapse free rate (1° outcome) and there was a reduction in contralateral testicular gem cell cancers (GCT). Because late relapses and 2nd GCT can occur beyond 5 and 10 yrs we have continued FU of these patients (pts). We report the updated results, and further analysis of C dose and outcome in relation to method of assessing renal function. Methods: The 1° randomisation was RT versus one injection of C dosed at 7× (GFR+25) based on EDTA(n=357) and 90% of this dose if based on creatinine clearance(n=202). The trial was powered to exclude absolute differences in the 2 yr relapse-free rates (RFR) of > 3%. RFR were determined by the Kaplan-Meier method, and hazard ratios (HR) from the Cox regression model; HR>1 favors RT. Results: Between 1996–2001, 1447 pts were randomised in a 3:5 ratio (C=573, RT=904). Median FU is now 6.5 yrs with documented minimum 5 yr FU on 1148 pts. RFRs at 5 yrs are 95% (C) and 96% (RT) (HR 1.25, 90% CI 0.83, 1.89); an increase in 5yr RFR of >3.6% can be excluded with 95% confidence. Only one death from seminoma (RT) has been reported. There was a significant difference in the rate of new GCTs (2 on C vs 15 on RT), giving a HR of 0.22 (95% CI 0.05, 0.95 p=0.03). High levels of pre-treatment FSH (>12 iu/L) were associated with an increased risk of developing a 2nd GCT (HR: 8.57 (95% CI 1.82 –40.38)) Analysing the variation in dose received in the C group, showed that those who received at least 99% of the 7AUC dose (n=347) ignoring GFR method had 5 yr RFR of 96.1% (95% CI 93.4–97.7) compared with 92.6% (88.0–95.5) in those who received lower doses (n=212, HR: 0.51 (0.24 - 1.07) p=0.08)). Analysis of the available data on pathological risk factors demonstrated that larger tumours (>4cm) had poorer RFR (HR: 3.68 (1.49 - 9.13)). Conclusion: With prolonged FU this trial confirms the non-inferiority of single dose C (AUC7) vs RT in terms of RFR but with a reduced risk of 2nd GCT in the C arm. The observation on the impact of C dosage, though not significant in its own right, reinforces the safety of adequately dosed C and may support studies of higher dose C. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Medical Research Council (non-commercial sponsor)

Estrogen: Roles in Spermatogenesis

In the mammalian testis, the irreversible conversion of androgens into estrogens is catalyzed by the cytochrome P450 aromatase, a microsomal enzymatic complex encoded by a unique gene (Cyp19) in humans which contained 18 exons, 9 of them being translated. In addition that gene includes 9 non-coding exons I, located in the 5 end and controlled by tissue-specific promoters that are spliced alternatively onto a common site in exon II. However, a unique protein of 55 kDa is produced. In most of mammals studied so far, at least in rodents, all testicular cells except peritubular cells contain aromatase which is therefore constitutively expressed and very precisely controlled (in terms of expression and enzyme activity) according to somatic and germ cell origin. In man, our data obtained from both ejaculated spermatozoa and immature gem cells demonstrate the presence of a biologically active aromatase ; moreover estrogen receptors (ER alpha and ER beta) are also present, especially ERβ in seminiferous tubules. All together according to the widespread localisation of aromatase and estrogen receptors in testicular cells, our review clearly shows that besides gonadotrophins and androgens, estrogens produced locally, should be considered as physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis. Therefore it is suggested that aromatase could be a helpful marker which might reflect the gene expression during spermatogenesis as well as the sperm quality and consequently, that parameter may be used as a diagnostic tool to assess the fertility in man. Keywords: Aromatase, Estrogens, Estrogen Receptors, Germ Cells, Spermatogenesis, Reproduction, Testis, Mammals

Production of germline chimeric chickens following the administration of a busulfan emulsion

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.

Mutations analysis of the HSP90 gene in infertile men with idiopathic azoospermia and severe oligozoospermia

Objective: Heat-shock proteins (HSP) are thought to act as molecular chaperones in variety of cellular process, which involve folding, transport, assembly and disassembly of polypeptide complexes. Several members of the HSP families are constitutively expressed during spermatogenesis, and are considered to be an essential factor for reproduction. HSP90 was expressed in gem cells, Sertoli cells, and Leydig cells during neonatal and early developing testis, and in spermatocytes and round spermatids after puberty (Cell Mol Life Sci; 1997, 53: 191–197). Scarce information exists on the role of sensitization to HSP and the potential role in the etiology of male infertility. In order to investigate whether mutations in the HSP90 gene are associate with severe subfertility men we screened polymorphisms in its coding region using SSCP (single strand conformation polymorphism) and DNA sequencing techniques from severe subfertile men.

Precocious puberty in children with tumours of the suprasellar and pineal areas: Organic central precocious puberty

During the past 11 y, 115 children younger than 8/9 y of age (female/male) with tumours of the suprasellar or pineal areas were followed in our clinic to study the incidence of precocious puberty. In addition, type of central lesion, clinical characteristics and gonadotropic secretion were studied in order to elucidate the different mechanisms of gonadal activation. A control group of 21 patients with idiopathic precocious puberty and a control group of 10 age-matched patients with suprasellar tumours without precocious puberty were also studied. Precocious puberty associated with organic central lesions was found at diagnosis in 30 patients (26%), in 9 out of 48 patients with glial cell tumours (18.7%), 6 out of 9 patients with gem cell tumours (66.6%), 11 out of 11 patients with hypothalamic hamartomas (100%) and in 4 out of 4 patients with subarachnoid cysts or arachnoidocele (100%). Precocious puberty was not found in any of 36 patients with craniopharyngioma. With the exception of one patient with pineal germinoma, all lesions were localized to the suprasellar area. In all patients with hypothalamic hamartoma, precocious puberty was diagnosed before 4 y of age, while in most patients with the other lesions, it was diagnosed after this age. Height SDS, weight increase and advancement of bone age were similar in both idiopathic and organic central precocious puberty. Maximal LH responses to GnRH in idiopathic and organic central precocious puberty were similar except for germ cell tumours. Patients with suprasellar tumours without precocious puberty had lower maximal LH (but not FSH) responses to GnRH, with the exception of germ cell tumours. In the latter, elevation of serum β-hCG indicates that this gonadotropin was responsible for gonadal stimulation. In hypothalamic hamartomas, the prepubertal hiatus in the activity of the GnRH pulse generator was absent. The mechanism of this failure in the inactivation of GnRH is unknown. Data suggest that in glial cell tumours and in subarachnoid cysts, an unknown factor, probably secreted by the tumours, advances the tempo of GnRH maturation. Therefore, the aetiology of organic central precocious puberty is multiple and is directly related to location and type of lesion. Conclusion: This clinical information suggests that the onset of puberty is not the result of the disruption of a putative pulse generator inhibitory influence but the consequence of secretion of stimulatory substances by the lesions.

Flex, an X-linked female-lethal mutation in Drosophila melanogaster controls the expression of Sex-lethal.

The Sex-lethal (Sxl) gene is required in Drosophila females for sexual differentiation of the soma, for gem cell differentiation and dosage compensation. We have isolated three new alleles of female-lethal-on-X (flex), an X-linked female-lethal mutation and have characterized its function in sex determination. SXL protein is missing in flex/flex embryos, however transcription from both Sxl(Pe), the early Sxl promoter and Sxl(Pm), the late maintenance promoter, is normal in flex homozygotes. In flex/flex embryos, Sxl mRNA is spliced in the male mode. Analysis of flex germline clones shows that it also functions in oogenesis, but in contrast to Sxl mutants that show an early arrest tumorous phenotype, flex mutant egg chambers develop to stage 10. In flex ovarian clones, Sxl RNA is also spliced in the male form. Hence, flex is a sex-specific regulator of Sxl functioning in both the soma and the germline. Genetic interaction studies show that flex does not enhance female lethality of Sxl loss-of-function alleles but it rescues the male-specific lethality of both of the gain-of-function Sxl mutations, Sxl(M1 )and Sxl(M4.) In contrast to mutations in splicing regulators of Sxl, the female lethality of flex is not rescued by either Sxl(M1 )or Sxl(M4). Based on these observations, we propose that flex regulates Sxl at a post-splicing stage and regulates either its translation or the stability of the SXL protein.

Associated leukemia and mixed gem cell tumor in a patient with gonadal dysgenesis

A 16-year-old phenotypic female developed acute myeloblastic leukemia with a fulminant course very shortly after surgery and chemotherapy for a mixed germ cell tumor of the ovary. The karyotype (46, XY, 47, XY + 8) suggested de novo rather than therapy-associated leukemia. The relationship between germ cell tumors and leukemia, their common yolk sac derivation and the role of the Y chromosome are discussed. The idea that XY gonadal dysgenesis may be familial also is discussed.