Environmental sustainability and the human element: Decision making in National Cancer Institute-designated cancer centers and Practice Greenhealth subscribing institutions

Background. Various aspects of sustainability have taken root in the hospital environment; however, decisions to pursue sustainable practices within the framework of a master plan are not fully developed in National Cancer Institute (NCI) -designated cancer centers and subscribing institutions to the Practice Greenhealth (PGH) listserv.^ Methods. This cross sectional study was designed to identify the organizational characteristics each study group pursed to implement sustainability practices, describe the barriers they encountered and reasons behind their choices for undertaking certain sustainability practices. A web-based questionnaire was pilot tested, and then sent out to 64 NCI-designated cancer centers and 1638 subscribing institutions to the PGH listserv.^ Results. Complete responses were received from 39 NCI-designated cancer centers and 58 subscribing institutions to the PGH listserv. NCI-designated cancer centers reported greater progress in integrating sustainability criteria into design and construction projects than hospitals of institutions subscribing to the PHG listserv (p-value = <0.05). Statistically significant differences were also identified between these two study groups in undertaking work life options, conducting energy usage assessments, developing energy conservation and optimization plans, implementing solid waste and hazardous waste minimization programs, using energy efficient vehicles and reporting sustainability progress to external stakeholders. NCI-designated cancer centers were further along in implementing these programs (p-value = <0.05). In comparing the self-identified NCI-designated cancer centers to centers that indicated they were both and NCI and PGH, the later had made greater progress in using their collective buying power to pursue sustainable purchasing practices within the medical community (p-value = <0.05). In both study groups, recycling programs were well developed.^ Conclusions. Employee involvement was viewed as the most important reason for both study groups to pursue recycling initiatives and incorporated environmental criteria into purchasing decisions. A written sustainability commitment did not readily translate into a high percentage that had developed a sustainability master plan. Coordination of sustainability programs through a designated sustainability professional was not being undertaken by a large number of institutions within each study group. This may be due to the current economic downturn or management's attention to the emerging health care legislation being debated in congress. ^ Lifecycle assessments, an element of a carbon footprint, are seen as emerging areas of opportunity for health care institutions that can be used to evaluate the total lifecycle costs of products and services.^

Exon involvement in the regulation of MuSVts110 RNA splicing

MuSVts110 is a conditionally defective mutant of Moloney murine sarcoma virus which undergoes a novel tmperature-dependent splice event at growth temperatures of 33$\sp\circ$C or lower. Relative to wild-type MuSV-124, MuSVts110 contains a 1487 base deletion spanning from the 3$\sp\prime$ end of the p30 gag coding region to just downstream of the first v-mos initiation codon. As a result, the gag and mos genes are fused out of frame and no v-mos protein is expressed. However, upon a shift to 33$\sp\circ$C or lower, a splice event occurs which removes 431 bases, realigns the gag and mos genes, and allows read-through translation of a P85gag-mos transforming protein. Interestingly, while the cryptic splice sites utilized in MuSVts110 are present and unaltered in MuSV-124, they are never used. Due to the 1487 base deletion, the MuSV-124 intron was reduced from 1919 to 431 bases suggesting that intron size might be involved in the activation of these cryptic splice sites in MuSVts110. Since the splicing phenotype of the MuSVts110 equivalent (TS32 DNA) which contains the identical 1487 base deletion introduced into otherwise wild-type MuSV-124 DNA, was indistinguishable from authentic MuSVts110, it was concluded that this deletion alone is responsible for activation of the cryptic splice sites used in MuSVts110. These results also confirmed that thermodependent splicing is an intrinsic property of the viral RNA and not due to some cellular defect. Furthermore, analysis of gag gene deletion and frameshift MuSVts110 mutants demonstrated that viral gag gene proteins do not play a role in regulation of MuSVts110 splicing. Instead, cis-acting viral sequences appear to mediate regulation of the splice event.^ Our initial observation that truncation of the MuSVts110 transcript, leaving only residual amounts of the flanking exon sequences, completely abolished splicing activity argued that exon sequences might participate in the regulation of the splice event.^ Analysis of exon sequence involvement has also identified cis-acting sequences important in the thermodependence of the splice event. Data suggest that regulation of the MuSVts110 splice event involves multiple interactions between specific intron and exon sequences and spliceosome components which together limit splicing activity to temperatures of 33$\sp\circ$C or lower while simultaneously restricting splicing to a maximum of 50% efficiency. (Abstract shortened with permission of author.) ^

Intron involvement in the regulation of MuSVts110 RNA splicing

The murine sarcoma virus MuSVts110 exhibits an alternative RNA splicing pattern. Like other simple retroviruses, MuSVts110 pre-mRNA splicing is balanced to allow the production of both spliced and unspliced RNA during the replicative cycle. In addition to balance, MuSVts110 RNA splicing exhibits a unique growth-temperature restriction to splicing; temperatures below 33$\sp\circ$C are permissive for splicing while temperatures of 37$\sp\circ$C or above are non-permissive. Previous work has established that this thermosensitive splicing phenotype is mediated in cis by viral transcript features. Here we show that at least three sequence elements regulate the MuSVts110 splicing phenotype. First, the MuSVts110 branchpoint (BP) and poly-pyrimidine tract (PPT) were found to be determinants of overall splicing efficiency. Wild-type MuSVts110 possesses a weak BP and PPT adjacent to the 3$\sp\prime$ splice site. Introduction of a strong BP caused MuSVts110 splicing to proceed to virtual completion in vivo, thus losing any vestige of balance or thermosensitivity. In in vitro splicing extracts, the strong BP overcame a blockade to wt MuSVts110 splicing at both the first and second catalytic steps. Weakening the consensus nature of the strong BP allowed the recovery of thermosensitive splicing in vivo, and reinstated the blockades to splicing in vitro, arguing that a suboptimal BP is an unusual manifestation of the proportional splicing pattern of retroviruses. The PPT is essential for accurate recognition of the BP sequence by the splicing machinery. Lengthening the PPT of MuSVts110 from 9 to 19 consecutive pyrimidines increased the overall efficiency of splicing in vivo dramatically, but was less effective than the strong BP in overriding the restriction on splicing imposed by high growth temperatures. Finally, decreasing gradually the overall size of the intron unexpectedly reduced splicing efficiency at growth temperatures permissive for splicing, suggesting that non-conserved sequences within the intron of MuSVts110 participate in splicing regulation as well. Taken together, these results suggest a mechanism of control in which MuSVts110 splicing is modulated by the entire intron, but principally by suboptimal signals at the splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing. ^

Cerebellar cortex involvement in classical eyelid conditioning

A model for cerebellar involvement in motor learning was tested using classical eyelid conditioning in the rabbit. Briefly, we assume that modifications of the strength of granule cell synapses at Purkinje cells in the cerebellar cortex and mossy fiber (MF) synapses at cerebellar interpositus nuclei are responsible for the acquisition, adaptively-timed expression, and extinction of conditioned eyelid responses (CRs). A corollary of these assumptions is that the cerebellar cortex is necessary for acquisition and extinction. This model also suggests a mechanism whereby the cerebellar cortex can discriminate different times during a conditioned stimulus (CS) and thus mediate the learned timing of CRs. Therefore, experiments were done to determine the role of the cerebellar cortex in the timing, extinction, and acquisition of CRs. Lesions of the cerebellar cortex that included the anterior lobe disrupted the learned timing of CRs such that they occurred at extremely short latencies. Stimulation of MFs in the middle cerebellar peduncle as the CS could support differently timed CRs in the same animal. These data indicate that synaptic plasticity in the cerebellar cortex mediates the learned timing of CRs. These short-latency CRs which resulted from anterior lobe damage did not extinguish, while CRs in animals receiving lesions which did not include the anterior lobe extinguished normally. Preliminary data suggests that lesions of the anterior lobe which produce short-latency responses prevent the acquisition of CRs to a novel CS. These findings indicate that the anterior lobe of cerebellar cortex is necessary for eyelid conditioning. The involvement of the anterior lobe in eyelid conditioning has not been previously reported, however, the anterior lobe has generally been spared in lesion studies examining cerebellar cortex involvement in eyelid conditioning due to its relatively inaccessible location. The observation that the anterior lobe of the cerebellar cortex is not always required for the basic expression of CRs, but is necessary for response timing, extinction, and acquisition, is consistent with the hypothesis that eyelid conditioning can involve plasticity in both the cerebellar cortex and interpositus nucleus and that plasticity in the nucleus is controlled by Purkinje cell activity. ^

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

Middle managers' attitudes toward employee assistance programs in the corporate setting

Several studies have shown that successful Employee Assistance Programs (EAPs) have strong management endorsement. Strong management endorsement is defined as positive support in utilizing EAP services for themselves and their employees. This study focuses solely on middle management as opposed to upper or general management support. The study further examines success or lack of success of an EAP by the utilization rate defined as the number of employees over a year period who access EAP services.^ A analytical cross-sectional design was used to compare and observe differences between two groups of middle managers (utilizers and nonutilizers). Middle manager data was collected through a mail questionnaire. The study focused on identifying predictors that influence middle managers' utilization rate specifically: attitude toward EAPs, EAP knowledge level, attitude toward mental health professionals, age, gender, years worked as a middle manager, education level, training, and other possible predictors of utilization. The overall hypothesis states middle manager utilizers of EAP services have more positive attitudes and a better understanding of their EAP than middle management nonutilizers.^ As predicted, nonparametric bivariate results showed significant differences between the two groups. Middle managers in the utilization group (n = 473) tended to show more positive attitudes toward their EAP and mental health professionals and demonstrated greater EAP knowledge compared to the nonutilization group (n = 154). These findings support past studies on variables that influence EAP utilization rates.^ Further variables found to influence middle management utilization were identified by multivariate logistic regression results. These variable were gender (female supervisors), educational levels of employees supervised (employees with lower levels of education), number of employees supervised (greater the number supervised, more likely to utilize), managerial EAP training (trained supervisors) and awareness that problems do influence an employee's productivity.^ These findings strengthen the assertion that middle management's attitudes, as well as other variables may influence utilization. Study findings add new information about important variables specifically influencing middle management who utilize EAPs. An understanding of these variables is essential in developing competent EAP program training and orientation programs for middle managers. ^

Evidence for the involvement of proximal chromosome 3P loci in the progression of Von Hippel-Lindau related tumors

Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder characterized by the development of retinal and central nervous system hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic islet cell tumors (PICT). The VHL gene maps to chromosome 3p25 and has been shown to be mutated in 57% of sporadic cases of RCC, implicating VHL in the genesis of RCC. We report a multigeneration VHL kindred in which four affected female siblings developed PICT at early ages. Analysis of the three coding exons of the VHL gene in this family revealed a single, missense mutation in codon 238. Inheritance of the 238 mutation has been reported to correlate with a 62% risk of pheochromocytoma development. In this kindred, all affected individuals carried the mutation as well as one additional sibling who showed no evidence of disease. Clinical screening of this individual indicated small ($<$1 cm) pancreatic and kidney tumors. Results suggest that inheritance of the codon 238 mutation does not correlate with early onset pheochromocytoma. Rather, the only individual in the pedigree with pheochromocytoma was the proband's mother who developed bilateral pheochromocytoma at the age of 62. Thus, the VHL codon 238 mutation may predispose to late onset pheochromocytoma in this family; however, it does not explain the preponderance of PICT in the third generation since this mutation has not been reported to increase the risk of developing pancreatic lesions. This suggests that inheritance of the codon 238 mutation and subsequent somatic inactivation of the wild type allele of the VHL gene may not be sufficient to explain the initiation and subsequent progression to malignancy in VHL-associated neoplasms. Since the two tumor types that most frequently progress to malignancy are RCC and PICT, we asked whether loss of heterozygosity (LOH) could be detected proximal to the VHL gene on chromosome 3 in distinct regions of 3p previously implicated by LOH and cytogenetic studies to contain tumor suppressor loci for RCC. LOH was performed on high molecular weight DNA isolated from peripheral blood and frozen tumor tissue of family members using microsatellite markers spanning 3p. Results indicated LOH for all informative 3p loci in tumor tissue from affected individuals with PICT. LOH was detected along the entire length of the chromosome arm and included the proximal region of 3p13-14.2 implicated in the hereditary form of renal cell carcinoma.^ If 3p LOH were a critical event in pancreatic islet cell tumorigenesis, then it should be expected that LOH in sporadic islet cell tumors would also be observed. We expanded LOH studies to include sporadic cases of PICT. Consistent LOH was observed on 3p with a highest frequency LOH in the region 3p21.2. This is the first evidence for an association between chromosome 3 loci and pancreatic islet cell tumorigenesis. (Abstract shortened by UMI.) ^

Involvement of a region of 23S ribosomal RNA of {\it E. coli\/} in decoding of termination codons

Involvement of E. coli 23S ribosomal RNA (rRNA) in decoding of termination codons was first indicated by the characterization of a 23S rRNA mutant that causes UGA-specific nonsense suppression. The work described here was begun to test the hypothesis that more 23S rRNA suppressors of specific nonsense mutations can be isolated and that they would occur non-randomly in the rRNA genes and be clustered in specific, functionally significant regions of rRNA.^ Approximately 2 kilobases of the gene for 23S rRNA were subjected to PCR random mutagenesis and the amplified products screened for suppression of nonsense mutations in trpA. All of the suppressor mutations obtained were located in a thirty-nucleotide part of the GTPase center, a conserved rRNA sequence and structure, and they and others made in that region by site-directed mutagenesis were shown to be UGA-specific in their suppression of termination codon mutations. These results proved the initial hypothesis and demonstrated that a group of nucleotides in this region are involved in decoding of the UGA termination codon. Further, it was shown that limitation of cellular availability or synthesis of L11, a ribosomal protein that binds to the GTPase center rRNA, resulted in suppression of termination codon mutations, suggesting the direct involvement of L11 in termination in vivo.^ Finally, in vivo analysis of certain site-specific mutations made in the GTPase center RNA demonstrated that (a) the G$\cdot$A base pair closing the hexanucleotide hairpin loop was not essential for normal termination, (b) the "U-turn" structure in the 1093 to 1098 hexaloop is critical for normal termination, (c) nucleotides A1095 and A1067, necessary for the binding to ribosomes of thiostrepton, an antibiotic that inhibits polypeptide release factor binding to ribosomes in vitro, are also necessary for normal peptide chain termination in vivo, and (d) involvement of this region of rRNA in termination is determined by some unique subset structure that includes particular nucleotides rather than merely by a general structural feature of the GTPase center.^ This work advances the understanding of peptide chain termination by demonstrating that the GTPase region of 23S rRNA participates in recognition of termination codons, through an associated ribosomal protein and specific conserved nucleotides and structural motifs in its RNA. ^

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

A MANAGEMENT INFORMATION SYSTEM AND EVALUATION SCHEME FOR THE UNIVERSITY OF TEXAS EMPLOYEE ASSISTANCE PROGRAMS

Early Employee Assistance Programs (EAPs) had their origin in humanitarian motives, and there was little concern for their cost/benefit ratios; however, as some programs began accumulating data and analyzing it over time, even with single variables such as absenteeism, it became apparent that the humanitarian reasons for a program could be reinforced by cost savings particularly when the existence of the program was subject to justification.^ Today there is general agreement that cost/benefit analyses of EAPs are desirable, but the specific models for such analyses, particularly those making use of sophisticated but simple computer based data management systems, are few.^ The purpose of this research and development project was to develop a method, a design, and a prototype for gathering managing and presenting information about EAPS. This scheme provides information retrieval and analyses relevant to such aspects of EAP operations as: (1) EAP personnel activities, (2) Supervisory training effectiveness, (3) Client population demographics, (4) Assessment and Referral Effectiveness, (5) Treatment network efficacy, (6) Economic worth of the EAP.^ This scheme has been implemented and made operational at The University of Texas Employee Assistance Programs for more than three years.^ Application of the scheme in the various programs has defined certain variables which remained necessary in all programs. Depending on the degree of aggressiveness for data acquisition maintained by program personnel, other program specific variables are also defined. ^

Involvement of histone H3 methylation in Tup1-Ssn6 repression

The Tup1-Ssn6 complex regulates the expression of diverse classes of genes in Saccharomyces cerevisiae including those regulated by mating type, DNA damage, glucose, and anaerobic stress. The complex is recruited to target genes by sequence-specific repressor proteins. Once recruited to particular promoters, it is not completely clear how it functions to block transcription. Repression probably occurs through interactions with both the basal transcriptional machinery and components of chromatin. Tup1 interactions with chromatin are strongly influenced by acetylation of histories H3 and H4. Tup1 binds to underacetylated histone tails and requires multiple histone deacetylases (HDACs) for its repressive functions. Like acetylation, histone methylation is involved in regulation of gene expression. The possible role of histone methylation in Tup1 repression is not known. Here we examined possible roles of histone methyltransferases in Tup1-Ssn6 functions. We found that like other genes, Tup1-Ssn6 target genes exhibit increases in the levels of histone H3 lysine 4 methylation upon activation. However, deletion of individual or multiple histone methyltransferases (HMTs) and other SET-domain containing genes has no apparent effect on Tup1-Ssn6 mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Since deletion of SET2 does not affect Tup1-Ssn6 repression, Ssn6 may utilize Set2 in other contexts to regulate gene repression. In order examine if the two components of the Tup1-Ssn6 complex have independent functions in the cell, we identified genes differentially expressed in tup1Δ and ssn6Δ mutants using DNA microarrays. Our data indicate that ∼4% of genes in the cell are regulated by Ssn6 independently of Tup1. In addition, expression of genes regulated by Tup1-Ssn6 seems to be differently affected by deletion of Ssn6 and deletion of Tup1, which indicates that these components might have separate functions. Our data shed new light on the classical view of Tup1-Ssn6 functions, and indicate that Ssn6 might have repressive functions as well. ^

Involvement of lipocalin 2 in leukemia and breast cancer

Human lipocalin 2 is described as the neutrophil gelatinase-associated lipocalin (NGAL). The lipocalin 2 gene encodes a small, secreted glycoprotein that possesses a variety of functions, of which the best characterized function is organic iron binding activity. Elevated NGAL expression has been observed in many human cancers including breast, colorectal, pancreatic and ovarian cancers. I focused on the characterization of NGAL function in chronic myelogenous leukemia (CML) and breast cancer. Using the leukemic xenograft mouse model, we demonstrated that over-expression of NGAL in K562 cells, a leukemic cell line, led to a higher apoptotic rate and an atrophy phenotype in the spleen of inoculated mice compared to K562 cells alone. These results indicate that NGAL plays a primary role in suppressing hematopoiesis by inducing apoptosis within normal hematopoietic cells. In the breast cancer project, we analyzed two microarray data sets of breast cancer cell lines ( n = 54) and primary breast cancer samples (n = 318), and demonstrated that high NGAL expression is significantly correlated with several tumor characteristics, including negative estrogen receptor (ER) status, positive HER2 status, high tumor grade, and lymph node metastasis. Ectopic NGAL expression in non-aggressive (ZR75.1 and MCF7) cells led to aggressive tumor phenotypes in vitro and in vivo. Conversely, knockdown of NGAL expression in various breast cancer cell lines by shRNA lentiviral infection significantly decreased migration, invasion, and metastasis activities of tumor cells both in vitro and in vivo . It has been previously reported that transgenic mice with a mutation in the region of trans-membrane domain (V664E) of HER2 develop mammary tumors that progress to lung metastasis. However, we observed that genetic deletion of the 24p3 gene, a mouse homolog of NGAL, in HER2 transgenic mice by breeding with 24p3-null mice resulted in a significant delay of mammary tumor formation and reduction of lung metastasis. Strikingly, we also found that treatment with affinity purified 24p3 antibodies in the 4T1 breast cancer mice strongly reduced lung metastasis. Our studies provide evidence that NGAL plays a critical role in breast cancer development and progression, and thus NGAL has potential as a new therapeutic target in breast cancer.^