Using MS-MLPA as an efficient screening tool for detecting 9p21 abnormalities in pediatric acute lymphoblastic leukemia.

BACKGROUND: Characterization of recurrent genetic lesions in childhood acute lymphoblastic leukemia (ALL) has enabled therapeutic stratification with improved outcomes. The tumor suppressor genes, CDKN2A and CDKN2B, encoding p16(INK4a) , p14(ARF) , and p15(INK4b) have been localized to 9p21. Abnormalities of 9p21 have been reported in 10-30% of childhood ALL using conventional cytogenetics and fluorescence in situ hybridization (FISH). The incidence of 9p21 using more sensitive techniques, such as methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), remains uncertain, and thus also the prognostic significance. PROCEDURE: We investigated the incidence and prognostic importance of 9p21 abnormalities in pediatric ALL patients using MS-MLPA and compared these results to FISH. RESULTS: In total, MS-MLPA or FISH detected aberrations (both dosage and methylation abnormalities) at 9p21 in a remarkable 32/48 (67%) patients in contrast to a much lower rate of only 8% of patients identified to have deletions by standard G banding cytogenetics. MS-MLPA identified five deletions not found by FISH. Aberrant methylation at CDKN2B was found in 19 (46%) patients. 9p21 abnormalities were associated with National Cancer Institute (NCI) high-risk criteria (P = 0.04) and were present in all five patients with T-cell disease. Four pre-B-cell ALL patients relapsed, three of whom had prior 9p21 abnormalities. CONCLUSIONS: MS-MLPA had a higher detection rate for 9p21 abnormalities than previously reported for other techniques. Given the ease of processing, minimal equipment and low cost of MS-MLPA, our results suggest that previous reports may have underestimated the true frequency of 9p21 abnormalities and their potential impact upon ALL outcome.

Resistin expression and regulation in mouse pituitary.

Resistin, a new adipocytokine, is expressed in human, rat and mouse adipose tissue. Its putative role as a mediator of insulin resistance is controversial. We hypothesized that resistin, in common with leptin, has multiple roles in non-adipose tissues. Using reverse transcription polymerase chain reaction (RT-PCR) we show that the resistin gene (Retn) is expressed in mouse brain (hypothalamus and cortex) and pituitary gland. Immunohistochemistry revealed resistin protein in the arcuate nucleus and pituitary gland. Semi-quantitative RT-PCR analysis indicated that Retn mRNA is developmentally regulated in the pituitary. Expression was lowest at birth, increased abruptly between postnatal days 14 and 25 (four-fold; P<0.001), and declined thereafter. This peak in pituitary Retn mRNA was unaffected by early weaning but was abolished by neonatal treatment with monosodium glutamate, suggesting that the basal hypothalamus regulates pituitary Retn. Although the role(s) of endogenous resistin in mouse brain and pituitary remains to be determined, it may be distinct from its controversial involvement in insulin resistance. Our data suggest that local resistin expression could have functional implications during prepubertal maturation of the hypothalamic-pituitary system.

Food restriction regulates adipose-specific cytokines in pituitary gland but not in hypothalamus.

White adipose tissue is now recognized as the source of a growing list of novel adipocyte-specific factors, or adipokines. These factors regulate energy homeostasis, including the response to food deprivation. We hypothesized that the brain and pituitary gland would also express adipokines and their regulatory factors and subsequently demonstrated that the rodent brain-pituitary system expresses mRNA and protein for leptin and resistin. We now report that the adipokines FIAF and adiponutrin, as well as the nuclear hormone receptor PPAR gamma, are expressed in pituitary, brain and adipose tissue. In pituitary gland, 24 h of food restriction reduced PPAR gamma expression by 54% whereas both adiponutrin and FIAF were increased 1.7 and 2.3 fold, respectively. These changes in expression were similar to those observed in fat, except for adiponutrin, which by contrast is dramatically reduced 95% by fasting. Furthermore, whereas PPAR gamma 2 is the main isoform affected by fasting in adipose tissue, our data suggest that only PPAR gamma 1 is present and downregulated by fasting in pituitary tissue. In contrast to the sensitivity of pituitary tissue to the effects of fasting, no significant change in expression was observed in basal hypothalamus for any of the genes studied. Overall, our data suggest that pituitary-derived adipokines may play an unexpected role in the neuroendocrine regulation of energy homeostasis.

Leptin receptors are developmentally regulated in rat pituitary and hypothalamus.

We have previously reported that leptin is expressed in adult rat brain and pituitary gland, though the role of leptin in these sites has not been determined. Leptin mRNA is developmentally regulated in the brain and pituitary of male and female rats during early postnatal development, suggesting a role in the maturation of the brain-pituitary system. Here, we sought to extend our previous studies by evaluating (1) the ontogeny of leptin receptor mRNA levels in rat brain and pituitary and (2) pituitary leptin protein levels in neonatal and pre-pubertal rats. Pituitary leptin concentration was highest shortly after birth (postnatal day (PD) 4, 25 ng/mg protein) and fell significantly throughout postnatal development and into adulthood (PD 60, 3.5 ng/mg protein; P<0.005) coincident with a decline in pituitary leptin mRNA levels. Significant age-related effects on leptin receptor mRNA levels were also observed in the pituitary and the hypothalamus of male and female rats using semi-quantitative RT-PCR analysis. In the pituitary, the short form (OBRa) mRNA levels were highest in neonatal rats (PD 4) but declined throughout postnatal development (PD 4-22) paralleling the fall in pituitary leptin mRNA and protein levels. The long form (OBRb) mRNA levels were unaffected by age between PD 4 and 22. In contrast, hypothalamic, levels of OBRb mRNA were very low to undetectable shortly after birth (PD 4) and rose significantly between PD 4 and 14/22 while levels of OBRa mRNA were not significantly different between PD 4 and 22. Immunohistochemical detection of leptin receptor immunoreactivity (all forms) revealed the presence of OBR-like protein in pituitary and hypothalamus as early as PD 4. Cortical leptin receptor mRNA levels were similar throughout early postnatal development. No gender-related differences in leptin receptor mRNA levels were noted in brain or pituitary. In conclusion, these data, together with our previous work, indicate that the neonatal pituitary gland expresses leptin and leptin receptors at levels far in excess of those observed in mature rats. The pituitary is thus quite different from adipose tissue, hypothalamus and cerebral cortex, in which neonatal leptin expression is lowest at birth. Since neonatal pituitary leptin receptor expression is also elevated, it is possible that pituitary-derived leptin plays some role in the development of the hypothalamic-pituitary system.

Leptin immunoreactivity is localized to neurons in rat brain.

Leptin is secreted from adipocytes and is thought to enter the brain to regulate and coordinate metabolism, feeding behaviour, energy balance and reproduction. It is now clear that there are many additional sites of leptin production, including human placenta, ovary, stomach, skeletal muscle, mammary gland, pituitary gland and brain. In the present work, we employed double-label immunofluorescent histochemistry to establish the neuronal localization of leptin immunoreactivity (IR). To accomplish this, we used the neuron-specific marker NeuN to label cells in the arcuate nucleus (ARC), piriform cortex and hippocampus. In the supraoptic nucleus (SON) and paraventricular nucleus (PVN), we used antisera to oxytocin and vasopressin as neuronal markers. Double labelling revealed leptin IR in neurons of the ARC and piriform cortex. Leptin IR was confined to the nucleus and to distinct perinuclear sites. In contrast, neurons in the CA 2/CA 3 region of the hippocampus showed little nuclear staining. Leptin IR was clustered around the nucleus in these cells. Neurons of the dentate gyrus exhibited both nuclear and perinuclear localization of leptin IR. In the SON/PVN, most oxytocin- and vasopressin-IR neurons also contained leptin IR, often in perinuclear sites. In conclusion, the neuronal, perinuclear localization of leptin IR in rat brain corresponds closely to that of leptin receptor (OB-R) IR, which has also been detected intracellularly. Our observation of leptin IR associated with cell nuclei suggests the existence of an OB-R distinct from the well-described membrane forms.

Pituitary resistin gene expression: effects of age, gender and obesity.

Resistin is a new adipocytokine which is expressed in rat, mouse and possibly human adipose tissue. Its putative role as a mediator of insulin resistance is controversial. We hypothesized that resistin, like leptin, would have multiple roles in non-adipose tissues and we reported that resistin is expressed in mouse brain and pituitary. Moreover, resistin expression in female mouse pituitary is developmentally regulated and maximal expression occurs peripubertally. Although the role of endogenous resistin in mouse brain and pituitary has not been determined, our data suggest that resistin could be important in the postnatal maturation of the hypothalamic-pituitary system. In the present study we compared the ontogeny of resistin gene expression in the pituitary of male and female mice using semi-quantitative RT-PCR analysis. We show that resistin expression is developmentally regulated in the pituitary of male and female CD1 mice. However, significant gender differences were evident (male > female at postnatal day 28 and 42) and this was not modified by neonatal treatment of female pups with testosterone. Since resistin expression in adipose tissue is also influenced by obesity, we evaluated resistin expression in fat, brain and pituitary of the obese ob/ob mouse. Resistin mRNA was significantly increased in both visceral and subcutaneous adipose depots in postnatal day 28 ob/ob mice compared to controls, but pituitary resistin expression was significantly reduced. In contrast to the prepubertal levels, and in agreement with other reports, adipose resistin expression was reduced in adult ob/ob mice. In a third set of experiments we examined the influence of food deprivation on pituitary and fat resistin mRNA. Resistin gene expression was severely down-regulated by a 24-hour fast in adipose and pituitary tissue but not in hypothalamus. In conclusion, pituitary resistin expression is age- and gender-dependent. In ob/ob mice, and in fasted mice, resistin is regulated in a tissue-specific manner. Thus in visceral fat obesity increases but starvation decreases resistin mRNA. In contrast, pituitary levels are decreased in the presence of both high (ob/ob) and low (fasting) adipose stores. Further studies are required to define the unexpected hormonal regulation of resistin gene expression in the pituitary.