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The PC cell line is a highly tumorigenic, insulin-independent, teratoma-derived cell line isolated from the nontumorigenic, insulin-dependent 1246 cell line. Studies of the PC cell growth properties have led to the purification of an 88-kDa secreted glycoprotein called PC cell-derived growth factor (PCDGF), which has been shown to stimulate the growth of PC cells as well as 3T3 fibroblasts. Sequencing of PCDGF cDNA demonstrated its identity to the precursor of a family of 6-kDa double-cysteine-rich polypeptides called epithelins or granulins (epithelin/granulin precursor). Since PCDGF was isolated from highly tumorigenic cells, its level of expression was examined in PC cells as well as in nontumorigenic and moderately tumorigenic cells from which PC cells were derived. Northern blot and Western blot analyses indicate that the levels of PCDGF mRNA and protein were very low in the nontumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic properties. Experiments were performed to determine whether the autocrine production of PCDGF was involved in the tumorigenicity of PC cells. For this purpose, we examined the in vivo growth properties in syngeneic C3H mice of PC cells where PCDGF expression had been inhibited by transfection of antisense PCDGF cDNA. The results show that inhibition of PCDGF expression resulted in a dramatic inhibition of tumorigenicity of the transfected cells when compared with empty-vector control cells. These data demonstrate the importance in tumor formation of overexpression of the novel growth factor PCDGF.

Previous studies have suggested that activation of calcium-independent PLA2 (CaIPLA2) is an early event in cell death after hypoxic injury in proximal tubule cells. An approximately 28-kD CaIPLA2 with preferential activity toward plasmalogen phospholipids has been recently purified from rabbit kidney cortex (D. Portilla and G. Dai, J Biol Chem 271, 15,451-15,457, 1996). Their report describes the cloning of a full-length rat cDNA encoding CaIPLA2, using sequences derived from the purified rabbit kidney cortex enzyme. In addition, cDNA from rabbit kidney that encode the rabbit homologue of the enzyme and a closely related isoform were isolated. The rat cDNA is predicted to encode an approximately 24-kD protein, and each cDNA contains the sequence G-F-S-Q-G, which fits the active site consensus sequence G-X-S-X-G of carboxylesterases. Several lines of evidence (DNA sequence comparison, Southern blot analysis, and examination of the expressed sequence tag database) show that CaIPLA2 enzymes are encoded by a multigene family in rats, mice, rabbits, and humans. Northern analysis of various tissues from the rat indicated that the CaIPLA2 gene is ubiquitously expressed, with highest mRNA abundance observed in the kidney and small intestine. The rat CaIPLA2 cDNA, when expressed in a baculovirus expression system, and the purified rabbit kidney cortex protein exhibit both CaIPLA2 and lysophospholipase activities. The cloned CaIPLA2 cDNA are expected to aid in understanding the role of CaIPLA2 in cell death after hypoxic/ischemic cell injury.

Adipose differentiation related protein (ADRP) is a 50 kDa protein expressed at high level in differentiated adipocytes. ADRP expression is very low in undifferentiated adipocytes and increases rapidly and dramatically as the cells undergo adipose differentiation. In the present study, we demonstrate that ADRP expression at the mRNA and protein level is stimulated in adipocyte precursor cells in a time- and dose-dependent fashion by treatment with cyclooxygenase inhibitors, particularly indomethacin and ibuprofen. Lipoxygenase inhibitors such as AA861 and nordihydroguaiaretic acid were ineffective. Stimulation of ADRP expression was observed with 10-5 M ibuprofen but maximal stimulation required a concentration of 3×10-4 M. Nuclear run-on experiments indicated that indomethacin or ibuprofen stimulated the transcription of the ADRP gene in undifferentiated adipocytes. In addition to stimulating the induction of ADRP in undifferentiated cells, ibuprofen and indomethacin also stimulated the level of ADRP mRNA and protein in differentiated adipocytes. These experiments provide new information on the regulation of ADRP, an early inducible gene in the adipocyte differentiation programme in adipocyte precursors and in adipocytes and identify a new target for cyclooxygenase inhibitor action during adipocyte differentiation.

Annually, about 220,000 patients are diagnosed with lung cancer in the US and 150,000 die of the disease. Advanced and recent therapeutic development in NSCLC addresses targets found in small percentages of patients, thereby emphasizing the need for continued discoveries of additional targets of lung cancer, in order to develop of novel therapies and diagnosis. We have investigated the role of the autocrine growth and survival factor GP88 (Progranulin) on the biology of Non-Small Cell Lung Carcinoma (NSCLC). Previously, GP88 had been shown to play a critical role in the proliferation and survival of breast cancer. Pathological studies with 600 cases of estrogen receptor positive invasive ductal carcinoma in correlation with clinical outcome data have also shown that GP88 tissue expression was associated with a 4-fold increased risk of recurrence and a 2.5-fold increase in mortality whereas inhibition of GP88 action by a neutralizing antibody resulted in inhibition of tumor formation of breast adenocarcinoma in vivo. In the present study, we examined the role of GP88 in NSCLC. Examination of GP88 protein expression by immunohistochemistry was carried out with formalin fixed paraffin embedded sections of lung cancer cases. Results showed that both human adenocarcinoma and squamous carcinoma stained positive for GP88 whereas normal lung tissues were negative. Pathological studies of tissue GP88 expression by IHC in 85 stage 1 and 2 NSCLC tumors in correlation with clinical outcomes demonstrated that high GP88 expression was associated with a 4-fold increase in the risk of recurrence (p=0.0004) indicating that GP88 is a prognostic factor for NSCLC. Based on these observations, expression and action of GP88 in NSCLC were examined using two representative human NSCLC cell lines A549 and H1299. Inhibition of GP88 expression by GP88 SiRNA resulted in an inhibition of cell migration and proliferation of these cells. Similarly, neutralization of GP88 action by treating the cells with AG1, an anti-human GP88 neutralizing monoclonal antibody expressed in recombinant form also inhibited cell proliferation and migration. Based on these results, studies were carried out to examine the effect of AG1 antibody on NSCLC tumor formation in nude mice. Preclinical studies show that AG1 treatment inhibits tumor formation by 30-50% when used as a single agent or in combination therapy with chemotherapeutic drugs used in the standard of care in NSCLC. These data to be presented here point out that GP88 is a novel therapeutic and prognostic target and provide new opportunities for targeted therapy with companion diagnostic for NSCLC. Citation Format: Ginette Serrero, Guanjun Xia, Jianping Dong, Binbin Yue, Amanda Schech, Douglas Hawkins, David Reisman, Pablo Bejarano, Jun Hayashi. GP88 (Progranulin) is a therapeutic and prognostic target for Non-Small Cell Lung Carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2013-2054

The 88 kDa glycoprotein GP88 (Progranulin, PCDGF, acrogranin) is the largest member of the granulin/epithelin family of growth modulators characterized by 7.5 cysteine-rich granulin/epithelin repeats. Our laboratory has demonstrated that GP88 represents an ideal therapeutic and diagnostic target in breast cancer. Our published studies have demonstrated that: 1) GP88 expression increases with tumorigenesis; 2) inhibition of GP88 expression by antisense transfection in MDA-MB-468 cells inhibited by 90% tumor formation in nude mice; 3) in ER+ breast cancer cells, GP88 stimulates proliferation and its overexpression confers estrogen independence and resistance to several anti-estrogens and aromatase inhibitor; 4) In Her-2 overexpressing breast tumors, GP88 stimulated Her-2 phosphorylation and conferred trastuzumab resistance; 5) GP88 is expressed in 80% invasive ductal carcinoma and 60% of ductal carcinoma whereas it is negative in lobular carcinoma, benign lesions and normal mammary epithelial cells; 6) pathological studies with 530 cases of ER+ invasive ductal carcinoma showed that GP88 tumor expression measured by immunohistochemistry (IHC) is a prognostic indicator of recurrence in early stage breast cancer patients; High GP88 tissue expression was associated with a 4-fold increase in risk of recurrence at 5 years; 7) GP88 is secreted and can be detected in the serum of breast cancer patients at an increased level when compared to healthy subjects. Based on these results, we developed a neutralizing anti-GP88 antibody AG1. AG1 was expressed in a high yield CHO cell line and formulated. AG1 inhibits GP88 biological effect (proliferation and migration) in a dose-dependent fashion in vitro. Here we will report the results of studies investigating the effect of AG1 on the tumor development of tamoxifen sensitive (MCF-7) and resistant (TamR) estrogen receptor positive breast cancer cells lines injected in nude mice. We show that AG1 alone (5mg/kg) inhibited tumor growth of MCF-7 cells injected into nude mice in a similar efficacy as tamoxifen. Interestingly, in tamoxifen resistant cells derived from MCF-7, the anti-GP88 antibody AG1 inhibited by more than 50% tumor formation of TamR cells in combination with tamoxifen, whereas tamoxifen only had no effect. These data suggest that inhibiting GP88 provides a novel and alternative therapeutic pathway for the restoration of tamoxifen sensitivity in ER+ breast cancer. This works is supported by grant 2R44CA124179 from the National Cancer Institute and 02–2010-010 from the Avon Foundation for Women. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-19.

Transforming growth factor-alpha (TGF alpha) and prostaglandin F2 alpha (PGF2 alpha) are potent inhibitors of adipocyte differentiation. We demonstrate here that TGF alpha messenger RNA (mRNA) is expressed in freshly isolated fat pads and in primary culture of adipocyte precursors cultivated in defined medium before and after differentiation. We show that PGF2 alpha stimulated TGF alpha mRNA expression in a dose-dependent manner. PGF2 alpha also stimulated TGF alpha production in the culture medium of adipocyte precursors in primary culture. PGF2 alpha stimulated TGF alpha mRNA expression in both undifferentiated and differentiated cells. 9 alpha,11 beta-PGF2 alpha, which also inhibited adipose differentiation, stimulated TGF alpha mRNA expression similarly to PGF2 alpha, whereas other PGs had no effect on TGF alpha mRNA expression. The time-course experiment indicates that the stimulation of TGF alpha mRNA expression by PGF2 alpha is observed within 6 h of exposure to PGF2 alpha and is inhibited by treatment of the cells with actinomycin D. The effect of PGF2 alpha on TGF alpha expression did not require activation of protein kinase C and was fully reversible. As both TGF alpha and PGF2 alpha are inhibitors of adipose differentiation, it is suggested that stimulation of TGF alpha expression by PGF2 alpha could represent an amplification mechanism to modulate adipocyte precursor differentiation and adipocyte function within the adipose tissue.

Prostaglandin F2 alpha (PGF2 alpha) and epidermal growth factor (EGF) are potent differentiation inhibitors of adipocyte precursors in primary culture. We show here that PGF2 alpha specifically inhibited EGF binding to adipocyte precursors in a dose dependent fashion. Scatchard analysis indicates that PGF2 alpha causes a 50% decrease in the number of available EGF cell surface receptors without change in receptor affinity. Comparison of EGF binding at different temperatures and on fixed cells indicates that PGF2 alpha increases internalization of EGF-receptor complexes in adipocyte precursors. Phorbol myristate acetate (PMA) also inhibited EGF binding in adipocyte precursors. PGF2 alpha effect was abolished in cells exposed to prolonged treatment with PMA indicating that PGF2 alpha effect on EGF binding is mediated by protein kinase C. These results would suggest that in adipocyte precursors PGF2 alpha may be the physiological mediator of phorbol ester effect on EGF receptor properties.

GP88 (Progranulin, PCDGF, acrogranin) is an 88 kDa glycoprotein that is the largest member of the granulin/epithelin family of growth modulators characterized by 7.5 repeats of a highly conserved 12-cysteine granulin/epithelin motif. Our laboratory demonstrated the role of GP88 as an autocrine growth and survival factor in breast cancer: 1) GP88 expression increases with tumorigenesis; 2) inhibition of GP88 expression by antisense transfection in MDA-MB-468 cells inhibited tumor formation in nude mice by 90%; 3) in ER+ breast cancer cells, GP88 stimulates proliferation and its overexpression confers estrogen independence; 4) GP88 is a strong survival factor that confers resistance to anti-estrogen and aromatase inhibitors in ER+ breast cancer; 5) GP88 is expressed in 80% invasive ductal carcinoma and 60% of ductal carcinoma whereas it is negative in benign lesions and normal mammary epithelial cells; 6) pathological studies with 530 cases of ER+ invasive ductal carcinoma showed that GP88 tumor expression is a prognostic indicator of recurrence in early stage breast cancer patients; 7) GP88 is secreted and can be detected in the serum of breast cancer patients at an increased level when compared to healthy subjects. Based on these results GP88 represents an ideal therapeutic and diagnostic target in breast cancer. We developed a neutralizing anti-GP88 antibody AG1. AG1 inhibits GP88 biological effect in a dose-dependent fashion. In the present study, we investigated the effect of AG1 on the tumor development of tamoxifen sensitive (MCF-7) and resistant (TamR) estrogen receptor positive breast cancer cells lines injected in nude mice. We show that AG1 alone (5mg/kg) inhibited tumor growth of MCF-7 cells injected into nude mice in a similar efficacy as tamoxifen. Interestingly, AG1 in combination with tamoxifen inhibited by more than 50% tumor formation of TamR cells, whereas tamoxifen only had no effect. These data suggest that inhibiting GP88 provide a novel and alternate pathway for restoration of tamoxifen sensitivity in ER+ breast cancer. This works is supported by grant 2R44CA124179 from the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2515. doi:1538-7445.AM2012-2515

This study was undertaken to compare the calcium-independent phospholipase A2 (PLA2) activities in the cytosols of twelve rat tissues and to determine whether their activities were distinct. 1-O-Alk-1'-enyl-2-[14C]-oleoyl-sn-glycero-3-phosphocholine (PlsC) and 1-O-Alk-1'-enyl-2-[14C]oleoyl-sn-glycero-3-phosphoethanolamine (PlsE) were synthesized and used as substrates, instead of phosphatidyl compounds, to exclude hydrolysis by cytosolic PLA1 activity that could be present in some of the cytosolic preparations. For each tissue, we examined substrate specificity, pH optimum, and effect of adenosine triphosphate (ATP) and ATP analogues. PLA2 activity was detected in eleven out of the twelve issues examined. Based on substrate specificity and pH optimum, cytosolic calcium-independent PLA2 were classified in three groups. The first group, which included PLA2 from small intestine, stomach and spleen, had the highest specific activity with PlsC as substrate (1253, 309 and 75 nmol/mg protein/hour, respectively) and an optimal pH at 6.5. Activity with PlsE as substrate was much lower (20-37%) than with PlsC. The second group of PLA2 activities included the cytosolic activities from thymus, lung, liver and pancreas that showed lower specific activities for both substrates (14-23 nmol/mg protein/hour with PlsC) and had a broader optimal pH range of 6.1 to 7.5. The cytosols from brain, kidney, heart and muscle comprised the third PLA2 group that was found to have a higher specific activity with PlsE (5-20 nmol/mg protein/hour) than PlsC and an optimal pH range from 7.4 to 7.9.(ABSTRACT TRUNCATED AT 250 WORDS)

Background Aromatase inhibitors (AI) that inhibit breast cancer cell growth by blocking estrogen synthesis have become the treatment of choice for post-menopausal women with estrogen receptor positive (ER+) breast cancer. However, some patients display de novo or acquired resistance to AI. Interactions between estrogen and growth factor signaling pathways have been identified in estrogen-responsive cells as one possible reason for acquisition of resistance. Our laboratory has characterized an autocrine growth factor overexpressed in invasive ductal carcinoma named PC-Cell Derived Growth Factor (GP88), also known as progranulin. In the present study, we investigated the role GP88 on the acquisition of resistance to letrozole in ER+ breast cancer cells Methods We used two aromatase overexpressing human breast cancer cell lines MCF-7-CA cells and AC1 cells and their letrozole resistant counterparts as study models. Effect of stimulating or inhibiting GP88 expression on proliferation, anchorage-independent growth, survival and letrozole responsiveness was examined. Results GP88 induced cell proliferation and conferred letrozole resistance in a time- and dose-dependent fashion. Conversely, naturally letrozole resistant breast cancer cells displayed a 10-fold increase in GP88 expression when compared to letrozole sensitive cells. GP88 overexpression, or exogenous addition blocked the inhibitory effect of letrozole on proliferation, and stimulated survival and soft agar colony formation. In letrozole resistant cells, silencing GP88 by siRNA inhibited cell proliferation and restored their sensitivity to letrozole. Conclusion Our findings provide information on the role of an alternate growth and survival factor on the acquisition of aromatase inhibitor resistance in ER+ breast cancer.

604 Background: GP88 (progranulin) is an autocrine growth factor produced by breast cancer cells in a positive correlation with tumorigenicity. It was shown that estrogen receptor positive (ER+) br...

Background: GP88 (progranulin) is a growth/survival factor previously characterized in our laboratory as a critical player of breast tumorigenesis and in the acquisition of resistance to anti-estrogen therapy in estrogen receptor positive (ER+) breast cancer. Previously reported pathological study showed that GP88 was preferentially expressed in ductal carcinoma rather than lobular carcinoma. THe highest expression was found in invaisve ductal carcinoma whereas normal mammmary tissue and benign lesions were negative. The present study examines the prognostic significance of GP88 and the association of GP88 expression with recurrence risk for breast cancer patients with ER+ invasive ductal carcinoma. Methods: Tissue GP88 expression was determined by immunohistochemistry using Oncostain 88 Kit on formalin-fixed paraffin embedded tissue sections followed by scoring by pathologists. Tumor GP88 expression was then correlated to survival outcomes in two retrospective multi-sites studies. The first training trial established the GP88 cut off value that is associated with decreased disease-free and overall survivals. The second study validated the GP88 cut off value and examined GP88 prognostic value compared to other prognostic factors in multivariate analysis. Results: The training trial demonstrated that GP88 expression was associated with a highly significant increase in the risk of recurrence of patients with ER+ invasive ductal carcinoma (IDC). This training trial established a GP88 cut-point where GP88 score of 3+ was associated with decreased disease-free and overall survivals. An independent validation study was performed with 264 distinct ER+ IDC cases to verify the strong impact of GP88 on survival outcomes and carry out multivariate analysis. The results confirmed the GP88 cut-point previously established with the training trial and showed that GP88 3+ was associated with a 4-fold higher hazard of disease recurrence when compared with GP88 < 3+ (GP88=0, 1+ or 2+). In addition, patients whose tumors had GP88=3+ showed a 2-fold higher mortality hazard when compared with the lower GP88 group. Multivariate analysis showed that GP88 was an independent risk predictor even after taking into account lymph node status, tumor size, tumor grade, and disease stage. Conclusion: The data show that the expression of the survival factor GP88 in breast tumor tissue is a prognostic factor, predictive of risk of recurrence and increased mortality for breast cancer patients with early stage ER+ IDC, independent from other prognostic markers. These results provide support for measuring GP88 tissue expression for newly diagnosed early stage breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2275. doi:10.1158/1538-7445.AM2011-2275

A cDNA clone referred to as 168 was previously isolated from mouse 1246 adipocytes by differential hybridization on the basis of its down regulation in adipocytes when compared to preadipocytes. 5' RACE was used to obtain a full length clone of 761 bp encoding for a highly basic 25 kD polypeptide that is extremely conserved in several diverse species of eukaryotes. There is a single amino acid substitution at position 202 compared to the human homolog, QM, a putative tumor suppressor. Clone 168 mRNA decreases 80% in rat primary culture of adipocytes compared to preadipocytes and does not decrease when differentiation is blocked by PGF2 alpha or EGF, indicating that the decrease is correlated with expression of the differentiation phenotype. Finally, two 1246 cell line variants that exhibit altered growth and increased tumorigenicity have a similar level of 168 mRNA when compared to the non tumorigenic adipogenic parent cell line.

Adipose differentiation-related protein (ADRP) is a novel 50-kDa membrane-associated protein whose message levels are induced rapidly and maximally after triggering adipocyte differentiation. The gene encoding mouse ADRP has been isolated and characterized from four overlapping λ phage clones. The gene spans 14 kb and contains 8 exons and 7 introns. Exons range in size from 50 to 696 bp and intron sizes range from 87 bp to 4.3 kb. Major and minor transcription initiation sites were determined 76 and 78 bp, respectively, upstream of the initiator methionine. A TATTTTA sequence is centered 30 bp upstream of the major transcription start site, and within the 5′-flanking region there are several putative transcription factor binding sites. ADRP has been mapped to chromosome 4, specifically between the b and Ifa loci. A second ADRP-like gene was isolated and partially characterized. This second locus is not expressed in 12 different mouse tissues and shares 87% sequence similarity to ADRP over exon and intron regions analyzed. Finally, this is the first reported genomic structure of ADRP.

PC cell line is a highly tumorigenic insulin-independent variant from the teratoma-derived adipogenic cell line 1246. Culture medium of PC cells contains a growth promoting activity for 3T3 cells and producer cells. PC cell-derived growth factor (PCDGF) was purified to homogeneity from PC cell-conditioned medium as an apparent 88-kDa protein by chromatography on heparin-Sepharose, Sephacryl S-200, and phenyl-Sepharose. Digestion with peptide-N-glycosidase F yielded an apparent 68-kDa protein component indicating that PCDGF is a glycoprotein containing about 20 kDa of carbohydrate. Partial sequence from Edman degradation of peptide fragments obtained by digestion of PCDGF with cyanogen bromide and trypsin demonstrates that PCDGF contains regions of sequence identity to that deduced from the granulin or epithelin precursor cDNAs. Granulins are small polypeptides purified from granulocyte extracts with no apparent biological functions. Epithelins are cell growth modulators purified as small molecular mass 6-kDa polypeptides from kidney extracts. The existence of a large molecular mass precursor for granulin or epithelin has been predicted based upon recently cloned cDNAs encoding these biomolecules within a 63.5-kDa protein with putative glycosylation sites. No biological activity has previously been attributed to the precursor. The present results indicate that PCDGF is a potential precursor for epithelin and/or granulin, that this 88-kDa protein is secreted and glycosylated, and that it can function as a mitogen for 3T3 cells as well as an autocrine growth factor for PC cells.

Epidermal growth factor (EGF) is a potent inhibitor of adipose differentiation in vitro and delays adipose tissue development in vivo. Here we show that in the homozygous male obese mice the level of EGF in the submaxillary gland and plasma is significantly lower than in the glands and plasma of age-matched control littermates. This EGF deficiency in ob/ob mice was observed as early as 5 wk of age when obesity had just become apparent and was also found in adult mice. The level of prepro-EGF mRNA expression in the submaxillary gland was also lower in obese mice than in control littermates. However, the level of kidney prepro-EGF mRNA was the same in mice with both phenotypes, suggesting that the regulation of prepro-EGF mRNA expression is different in both tissues. These results indicate that genetic obesity in mice is accompanied by a decrease in the production of EGF.

Obesity which is characterized by abnormal adipose tissue development is a first degree public health hazard in industrialized countries as it is accompanied by a high incidence of cardiovascular disease, diabetes and elevated morbidity. One important aspect in the study of adipose tissue development is to investigate the hormonal control of adipose differentiation. In particular, if negative regulators of differentiation can be identified, than they can be used to develop a therapeutic approach towards the control of excessive adipose tissue development in human subjects. The data presented here describe the identification of epidermal growth factor as a potent inhibitor of adipose differentiation both in vitro and in vivo. Moreover, evidence are presented that genetically obese ob/ob mice present an impaired level of EGF thus demonstrating for the first time the existence of a correlation between low level of the adipose differentiation inhibitor EGF and excessive adipose tissue development.

The isolation of a cDNA corresponding to a portion (amino acid 943 to 1073) of the cytoplasmic domain of the mouse EGF receptor surrounding the auto phosphorylation sites was obtained by using the reverse transcriptase polymerase chain reaction (RT-PCR) approach. Deduced amino acid sequence of mouse EGF receptor (EGFr) shows a 92% and 76% homology to corresponding regions in the human and the chicken EGFr, respectively. This cDNA was used to develop a sensitive RNase protection assay to investigate EGF receptor mRNA expression in mouse C3H teratoma derived cell lines with increased tumorigenic properties which display a progressive decrease of EGF binding and response. The results show that increased tumorigenicity was not accompanied by a change in EGF receptor mRNA expression. Moreover, they indicate that the RNase protection assay developed using the probe described here is a sensitive approach to investigate EGF receptor expression in murine cells.

e18103 Background: GP88, is an 88 kDa glycoprotein autocrine growth factor belonging to a newly identified family of growth modulators characterized by unique cysteine-rich motif and playing a role...

In 2009, 219,000 patients were diagnosed with lung cancer in the US and 159,000 died of the disease. It has been widely reported that results of standard treatment in lung cancer are poor except for the most localized cancers, thereby emphasizing the need to discover novel targets of lung cancer for the development of novel therapies and diagnosis. In the present study, we investigated the role of the autocrine growth and survival factor GP88. GP88 (also known as Progranulin) is an 88kDa glycoprotein that has been shown to play a critical role in the proliferation and survival of breast cancer. Pathological studies with breast cancer cases in correlation with clinical outcome data have also shown that GP88 expression was associated in invasive ductal carcinoma with increased risk of recurrence. In the present study, we examined the role of GP88 in non small cell lung carcinoma (NSCLC). Examination of GP88 protein expression by immunohistochemistry using Oncostain 88 kit was carried out with formalin fixed paraffin embedded sections of lung cancer cases. Results showed that non small cell lung carcinoma, adenocarcinoma and squamous carcinoma stained positive for GP88 whereas small cell carcinoma sections were mostly negative. In addition, normal lung tissues did not express GP88. Based on this observation, expression and action of GP88 in NSCLC was examined using representative cell lines. We show here that GP88 stimulated proliferation and survival of A549 and H1299 cell lines. GP88 expression was upregulated in cells that were made resistant to cytotoxic drugs such as taxol and velcade. Inhibition of GP88 expression by SiRNA or GP88 action by treating the cells with AG1, an anti-GP88 monoclonal antibody resulted in an inhibition of cell migration and proliferation of these drug resistant cells and increased response to cytotoxic drugs. Based on these results, studies were carried out to examine the effect of AG1 antibody on migration and tumor formation of A549 and H1299 cells in vitro and in vivo. Our data that will be presented here point out that GP88 is a novel therapeutic target for NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4113.

e22021 Background: The autocrine growth factor GP88 is an important player in breast cancer. GP88 is expressed in human BC tumors in correlation with their tumorigenicity. Increased GP88 expression was associated with anti-estrogen therapy resistance in ER+ cells and Herceptin resistance in Her-2 overexpressing breast tumors. Inhibition of GP88 expression inhibited tumor incidence and growth in nude mice. Immunohistochemical studies have shown that GP88 is expressed in invasive ductal carcinomas (IDC) and that high GP88 expression correlated with increased recurrence and mortality. Since GP88 is found in serum, we hypothesized that GP88 was elevated in the sera of breast cancer patients compared to healthy individuals and that GP88 serum level increases with disease progression. Methods: An IRB approved prospective study was established at the University of Maryland Breast Clinic to determine the serum level of GP88 in breast cancer patients (BC pts). Approximately 5 ml of blood was drawn every three months. GP88 serum concentration was determined in triplicate by human GP88 enzyme immunoassay. 190 BC pts were accrued. Sera from healthy volunteers (HV) were obtained to establish GP88 baseline. BC patient characteristics: Caucasian- 91, African American-92, Asian-6; median age, 51 (range 29- 86), stage I - 48, II - 52, III - 26, IV - 63. Results: Median serum GP88 level was 28.7 ng/ml (range 16.6–38.2) in HV, 40.7 ng/ml (range 6.4–100) in early stage (stage 1 -3) BC pts (p- value = 0.007) and 45.3 ng/ml (range 9.8 to 158.4) in stage 4 BC patients (p- value= 0.0007). Statistically significant increase in serum GP88 level was found in early stages as well as in metastatic disease when compared to HV. In addition, patients that were initially diagnosed with early stage disease but recurred showed a 5 to 10 fold increase in their GP88 serum levels. Conclusions: GP88 serum level is significantly higher in the sera of BC than HV subjects. Moreover, GP88 serum level increased in association with disease recurrence and progression. This study identifies GP88 as a measurable biomarker for disease progression not only at the tissue but also at the serum level. These results are also interesting since GP88 is also a therapeutic target of malignant progression of breast carcinoma. No significant financial relationships to disclose.

Abstract #2006 Background: GP88 is an autocrine growth factor that plays a critical role in breast tumorigenesis. GP88 is expressed in human BC tumors in a positive correlation with their tumorigenicity. Increased GP88 expression is associated with resistance to anti-estrogen therapy in ER + cells and with herceptin resistance in Her-2 overexpressing breast tumors. Inhibition of GP88 expression in human breast adenocarcinoma inhibited tumor incidence and growth in nude mice. Immunohistochemical studies have shown that GP88 was expressed in invasive ductal carcinomas in correlation with the expression of poor prognosis markers whereas normal tissues and benign lesions were negative. High GP88 expression in tumor biopsies was accompanied by decreased disease-free survival. Since GP88 can be secreted, we have hypothesized that GP88 could be secreted in the circulation and found in serum. We examined whether GP88 could be found in the circulation and whether GP88 could be elevated in the sera of breast cancer patients when compared to healthy individuals. Methods: An IRB approved blood sampling study was conducted at the University of Maryland Breast Clinic to determine the serum level of GP88 in healthy volunteers (HV) and breast cancer patients (BC pts). Serum GP88 concentration was determined in triplicate by quantitative enzyme immunoassay. 189 BC pts were accrued. In addition, sera from 18 HV were obtained to establish a GP88 baseline in healthy volunteers. BC patient characteristics: Race: Caucasian- 91, African American-92, Asian-6; median age, 51 (range 29-86), stage I – 48, II - 52, III – 26, IV - 63. Results: Circulating GP88 was measurable in the serum. Median level of GP88 was 28.7 ng/ml (range 16.6-38.2) in HV; 40.7 ng/ml (range 6.4-100) in early stage (stage 1 –3) BC pts (p- value = 0.007) and 45.3 ng/ml (range 9.8 to 158.4) in stage 4 metastatic BC patients (p-value= 0.0007). Statistically significant increase in circulating GP88 level was found in early stages as well as in metastatic disease. Correlation studies with BC prognostic factors such as stage, tumor size, lymph node involvement, tumor grade and presence of ER and HER-2 will be presented. Conclusion: GP88 can be detected in the sera of HV and BC pts. Comparison between the two groups of subjects indicates that GP88 level is significantly higher in the sera of BC pts. These studies are important as they identify as a measurable circulating biomarker GP88 that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC). Future studies will examine whether there is any correlation between the serum level of GP88 and therapeutic response to systemic therapy in breast cancer patients. This study was supported by grant from MIPS, the Avon Foundation and 1R43 CA 124179-01A1 from the National Institutes of Health. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2006.

Abstract #1088 Background Our laboratory is working on the characterization of novel breast cancer targets that have therapeutic an/or diagnostic applications. We have previously identified the growth factor GP88 as a significant player of breast tumorigenesis. GP88 is expressed in human breast cancer cells in a positive correlation with tumorigenesis. In estrogen receptor positive breast cancer cells, GP88 mediates estrogen proliferation effect and its increase is associated with resistance to anti-estrogen therapy. Inhibition of GP88 expression in human breast adenocarcinoma lead to inhibition of tumor incidence and tumor growth in nude mice. Pathological studies showed that ductal carcinoma expressed GP88 whereas benign lesions and normal mammary tissue were negative. Based on this evidence, the present study was carried out to investigate whether high GP88 expression in estrogen receptor positive invasive ductal carcinoma is associated with increased recurrence and decreased overall survival. Methods Under an IRB approved study, 239 archival formalin-fixed, paraffin-embedded estrogen receptor positive invasive ductal carcinoma with a minimum of 4 years follow-up were obtained from three United States tumor banks The clinical data included patients demographics such as age and race, tumor characteristics including tumor size, tumor grade, nodal status, disease stage and receptor status, time and type of first recurrence, time and status of last follow-up. GP88 expression was determined by immunohistochemistry using Oncostain 88TM IHC kit on five-micrometer sections. GP88 expression was scored as: 10% of cells staining – positive with positive staining graded from weak/focal (1+) to moderate/focal or diffuse (2+) to strong/diffuse (3+). The statistical analysis of GP88 expression in all cases was carried out. The Oncostain 88TM test performance was evaluated for its ability to predict disease-free (DFS) and/or overall survivals (OS) using Kaplan Meier curves and the Cox proportional hazards models for quantification of risk. Results The data show that ER+ cases with high GP88 expression (3+) had a hazard ratio for OS of 2.29 (95% CI, 1.38-3.78; p< 0.0013) and a HR for DFS of 2.88 (95% CI 1.59-5.24; p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1088.

22064 Background: Our laboratory focuses on the development of novel breast cancer biomarker with diagnostic or prognostic significance. We have identified a growth factor GP88 that plays a critica...

551 Background: The identification of a measurable biomarker that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC) is needed. PCDGF/GP88, a...

A heat-sensitive, trypsin-sensitive factor that promoted growth and spreading of cells in serum-free, hormone-supplemented medium was partially purified from human serum. The major portion of the proteins in these preparations migrated upon SDS-polyacrylamide gel electrophoresis with a mobility consistent with molecular weights between 60,000 and 90,000. The spreading activity, which we have termed serum spreading factor, stimulated growth and spreading of a wide variety of cell types. The serum spreading factor was similar to fibronectin in that it showed an affinity for the plastic cell culture substrate but was shown to be distinct from fibronectin by several criteria. This factor may prove useful in studies of cell attachment and spreading and in studies of the relationship of cell shape and cell proliferation.

The 1246-3A cell line is an insulin-independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246-3A cell line releases in its culture medium two types of transforming growth factors, TGF-alpha- and TGF-beta-like polypeptides, and a growth inhibitor. TGF-alpha like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high-molecular-weight TGF-alpha-like factors competed with 125I-EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF-alpha antibody, not by incubation with anti-EGF antibody. Both fractions promoted anchorage-independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c-3T3 cells in a fashion similar to EGF and synthetic TGF-alpha. In addition to secreting TGF-alpha-like polypeptides, 1246-3A cells produce TGF-beta. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage-independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5-11 kDa. This fraction was different from TGF-beta and TGF-alpha as determined by specific radioreceptor competition assays. TGF-alpha and TGF-beta-like polypeptides could represent autocrine growth stimulators for the insulin-independent 1246-3A cells and act in synergy with insulin-related factor (IRF) for an optimal stimulation of 1246-3A cell proliferation in serum-free medium.

Adipose differentiation was studied in a teratoma-derived fibroadipogenic cell line (1246) cultured in serum-free medium. The addition of dexamethasone and 1-methyl-3-isobutylxanthine to the serum-free medium induced confluent 1246 cells to differentiate into adipocyte-like cells as evidenced by triglyceride accumulation and increased levels of lipolytic enzyme activities. Hormone-sensitive lipase activity measured 5 days after the addition of dexamethasone and 1-methyl-3-isobutylxanthine increased 17-fold and was activated by cAMP-dependent protein kinase. Neutral diglyceride lipase, monoglyceride lipase, and cholesterol ester hydrolase specific activities increased 23-, 75-, and 73-fold, respectively. Among these three activities, only cholesterol ester hydrolase was activated by cAMP-dependent protein kinase. Differentiated 1246 cells expressed receptors to lipolytic hormones as shown by the stimulation of glycerol release by epinephrine (8.6-fold), glucagon (2.2-fold), and adrenocorticotrophic hormone (5.5-fold). Heparin treatment of 1246 cells in serum-free medium resulted in the release of lipoprotein lipase activity into the culture medium. Thus, 1246 cells can serve as a model for the study of adipose differentiation under defined culture conditions since they are capable of growth and survival in the absence of serum while retaining their ability to differentiate into adipocytes.

This paper describes a method to establish primary and secondary cultures of adipocyte precursors isolated from inguinal fat pads of 48-hr-old rats in defined medium. The culture medium consists of DME-F12 medium supplemented with fibronectin, insulin, transferrin, and fibroblast growth factor. Data presented indicate that 90% of the cells plated in the 4F medium differentiate in 7 d. These cultures provide appropriate differentiation assay systems to characterize regulators of differentiation acting on normal cells derived from the adipose tissue.

The adipogenic cell line 1246, which grows and differentiates in defined medium, stringently requires insulin for both processes. From this cell line, insulin-independent variants were isolated and characterized. Unlike 1246 cells, the variant cell lines proliferate without insulin, have lost their differentiation ability, produce factor(s) able to replace insulin to stimulate 1246 cell growth but not differentiation and are tumorigenic. Because of these properties, this system is appropriate to examine the correlation (if any) between the loss of response to an extra-cellular factor and of ability to differentiate, and between the production of endogenous growth factor and the acquisition of tumorigenic properties.

Newborn rat adipocyte precursors, isolated from inguinal fat pads of 2 day-old NBR rats proliferate and undergo adipose differentiation in defined medium in the absence of serum when cultivated on polylysine coated dishes in DME-F12 medium supplemented with fibronectin, insulin, transferrin and FGF. After 7 days in culture in these conditions, 90% of the cells have undergone differentiation as measured by the increase of G3PDH specific activity and by the accumulation of triglycerides in their cytoplasm. In contrast, the cells cultivated in the presence of 10% fetal bovine serum, have a limited ability to differentiate. These results indicate that newborn rat adipocyte precursors from inguinal fat pads do not require the presence of an undefined adipogenic factor in order to differentiate in culture. In contrast, proliferation and differentiation are dependent on the presence of insulin in the culture medium. Moreover, the data presented in this paper show that the rat adipocyte precursor culture represents a rapid and reproducible system for investigating the processes of adipose tissue development and for studying the negative and positive regulators of the adipose differentiation in a controlled environment.

An insulin-independent teratoma-derived cell line, called 1246-3A, has been isolated from the adipogenic cell line 1246, which stringently requires insulin for proliferation. The 1246-3A cell line, which can proliferate in the absence of exogenous insulin, produces in its conditioned medium a growth factor similar to pancreatic insulin by its biological and immunological properties. This factor, called "insulin-related factor" (IRF), was purified and iodinated to study its binding to cell surface receptors. 125I-labeled IRF binding to intact 1246-3A cells is lower than to 1246 cells. Cell surface binding can be restored by culturing the 1246-3A cells in the presence of an anti-porcine insulin monoclonal antibody or by acid prewash of the cells prior to performing the binding. Scatchard analysis of binding indicates that IRF secreted by the 1246-3A cells partially occupies high-affinity binding sites on the producer cells. Moreover, insulin monoclonal antibody inhibits the proliferation of the IRF-producing 1246-3A cells, suggesting that these cells are dependent on the secreted IRF for growth in culture. We conclude that the insulin-related factor secreted by the insulin-independent 1246-3A cells stimulates their proliferation in an autocrine fashion.

Recent studies indicate that lipid droplets isolated from a variety of different cells are rich in proteins known to regulate membrane traffic. Among these proteins are multiple Rab GTPases. Rabs are GTP switches that regulate intracellular membrane traffic through an ability to control membrane–membrane docking as well as vesicle motility. Here we present evidence that the multiple Rabs associated with droplets have a function in regulating membrane traffic. Droplet Rabs are removed by Rab GDP-dissociation inhibitor (RabGDI) in a GDP-dependent reaction, and are recruited to Rab-depleted droplets from cytosol in a GTP-dependent reaction. Rabs also control the recruitment of the early endosome (EE) marker EEA1 from cytosol. We use an in vitro reconstitution assay to show that transferrin receptor positive EEs bind to the droplet in a GTP/Rab-dependent reaction that appears not to lead to membrane fusion. This docking reaction is insensitive to ATPγs but is blocked by ATP. Finally, we show that when GTP bound active or GDP bound inactive Rab5 is targeted to the droplet, the active form recruits EEA1. We conclude that the Rabs associated with droplets may be capable of regulating the transient interaction of specific membrane systems, probably to transport lipids between membrane compartments.

The hydrolysis of trioctanoin and of monoolein by rat intestinal homogenates, catalyzed by the previously described "trioctanoin hydrolase" and "monoglyceride lipase" [Ref. 6 and 11], was shown to be entirely due to the action of the intestinal glycerol-ester hydrolase [Ref. 1 and 2] and to contaminating pancreatic lipase firmly bound to the intestinal cells. These conclusions are supported by experiments based on the chromatographic behaviour of the different lipolytic activities, the use of an immunoserum directed against rat pancreatic juice and the relief of bile salt inhibitions by pancreatic colipase. The results are in favour of the existence of a single lipolytic enzyme in the rat intestinal mucosa, i.e. glycerol-ester hydrolase, active against short- and medium chain mono-, di- and triglycerides and against long chain monoglycerides.

Publisher Summary This chapter presents the practical details for the growth of cells in serum free hormone supplemented media. In the absence of serum, greater than usual care must be taken in preparation of the synthetic portion of the medium. Careful preparation of water is essential for consistent results with serum-free medium. Serum and even dialyzed serum can mask nutritional requirements of cells in culture. Commercially available powdered media are adequate for serum-free work although each batch must be checked for suitability. Serum also serves the function of a trypsin inhibitor in conventional tissue culture procedures. The serum may be necessary for the repair of trypsinization damage after subculture; or the residual serum left after its removal, even after one or more washes, may be furnishing unknown growth factors.

Adipocyte precursors, isolated from inguinal fat pads of 2-day old NBR rats, proliferate and differentiate in defined medium. EGF stimulates the proliferation of adipocyte precursors with an ED50 of 2 ng/ml and inhibits their differentiation with an ED50 of 0.45 ng/ml. EGF has no effect on differentiated adipocytes. EGF binding studies indicate that adipocyte precursors have two classes of receptors (Kd 4 × 10−11 M, 4800 receptors/cell and Kd 1.4 × 10−9 M, 22,000 receptors/cell). TGF-α has the same effect as EGF on adipocyte precursors. In contrast, basic and acidic FGF stimulate adipocyte precursor proliferation without inhibiting their differentiation.

Adipose differentiation has been actively studied since the isolation and establishment in culture of several triglyceride-producing cell lines which provide useful in vitro models (1–6). It is now possible to investigate various problems related to the process of differentiation, particularly the determination of the nature and role of extracellular factors controlling the growth and the differentiation of adipocyte-like cells. Since serum-free culture has been successfully done with a number of cell lines for the past few years (7), one possible approach to the latter problem is to cultivate adipocyte-1ike cells in a serum-free medium. If the identities of the factors supporting adipocyte growth and differentiation are known, understanding their function might be possible. Attempts were made to cultivate, in serum-free medium, three different adipocyte-1ike cell lines. There were 3T3-L1 (8), Ob17 (Gaillard et al, in preparation) and 1246 (6). 3T3-L1 cell line is derived from Swiss mouse 3T3 fibroblasts (10; Ob17 cell line has been isolated from the epididymal fat pad of C57B16J Ob/Ob mouse (3); 1246 cell line has been clonally derived from a cell line, C17-S1-D-T984, isolated from a teratoma obtained by injecting an embryonal carcinoma cell line C17-S1 into a C3H mouse (6,9). It was found that 3T3-L1 and Ob17 cell lines have the same growth requirements in serum-free medium, i.e., fibronectin, insulin, transferrin, fibroblast growth factor (FGF) and a factor isolated from female rat submaxillary gland extracts (8,10). The 1246 cell line only requires fibronectin, insulin, transferrin and FGF, and does not need the presence of the submaxillary gland factor (6).

1246-3A cell line is an insulin-independent variant isolated from the adipogenic cell line 1246 which can proliferate in the absence of insulin, has lost the ability to differentiate, and secretes an insulin-related factor called IRF similar to pancreatic insulin and different from IGFs. In contrast, the parent adipogenic cell line 1246 is dependent on the presence of insulin to proliferate and differentiate in defined medium. In the present paper, we examined if the loss of response to insulin observed for 1246-3A cells was accompanied by alterations in the insulin receptor properties. Insulin binding and tyrosine kinase activity of insulin receptors isolated from 1246-3A cells and from the parent cell line 1246 were measured; 125I-insulin binding to intact cells was 75% lower for the 1246-3A cells than for the 1246 cells. This was due to a decrease in receptor number without major change in receptor affinity. However, when the cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed for both cell lines. Down regulation of insulin receptors by insulin occurred in a dose-dependent fashion, which was similar for both cell lines. Phosphorylation experiments were performed by incubation of the partially purified insulin receptor with insulin and [gamma-32P]ATP. They indicated that insulin stimulated phosphorylation of the 95-kDa molecular weight beta subunit of the receptor, in a similar fashion for both cell types. These data suggest that the insulin-independent cell line 1246-3A does not possess a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties and that the loss of response to insulin can be attributed to the fact the 1246-3A cells secrete IRF which bind to cell surface receptors and stimulate their proliferation.

C17-S1-D-T984 (to be referred to as T984) is a myogenic clonal cell line isolated from a mouse teratocarcinoma. T984 exhibits phenotypic instability since it gives rise not only to myogenic but also to fibro-adipogenic and fibroblastic clones. A cell line of each clone type has been established and studied with respect to (1) phenotypic expression and stability; and (2) growth and differentiation in serum-free and serum-supplemented media. In both respects, marked differences between the three cell lines were observed. All three cell lines respond by increased growth in serum-free media to insulin, transferrin, fibroblast growth factor (FGF) and the serum-spreading factor of Holmes. The fibroblastic and the fibro-adipogenic cell lines can both be grown indefinitely in a serum-free medium which contains the above factors. The fibro-adipogenic cell line, which differentiates in serum-supplemented medium, exhibits very limited differentiation in the absence of serum; the serum factor(s) required for adipogenic differentiation is (are) probably proteins of molecular weight superior to 10 000. In direct contrast, the myogenic cell line exhibits limited growth in serum-free medium but readily differentiates under these conditions. Moreover, myogenic differentiation could be obtained in the defined medium at very low densities and was not influenced by the addition of medium conditioned by cells seeded at high densities. Thus, in this system, muscular differentiation is apparently independent of diffusible endogenous or exogenous factors and is probably triggered by the arrest of growth. While our results do not explain the reason why T984 exhibits phenotypic instability, they do indicate that this clonal cell line and its clonal derivatives could be used to identify the factors that influence the growth and the differentiation of cells of different mesenchymal phenotypes. The possible relationship of phenotypic instability to muscular dystrophies is also discussed.

The teratoma-derived insulin-independent cell line 1246-3A produces and secretes polypeptide mitogens in its culture serum-free medium. Mitogenic activities were separated by Sephadex G-50 chromatography into two fractions eluted, one in the void volume region, another one with an apparent Mr of 6 kDa. Only the 6 kDa mitogen presents properties similar to pancreatic insulin as estimated by radioimmunoassay, radio-receptor assay, and biochemical characterization. As a consequence, this factor is called insulin-related factor (IRF). Evidence presented in this paper indicate that ectopic IRF binds to insulin receptors on the producer cells, 1246-3A and acts in an autostimulatory manner.

Obesity arises from an abnormal growth and differentiation of adipose cells. Previous works have shown that obesity could be due to either an increase in adipose cell number (hyperplasia) or an increase in the triglyceride content of adipocytes (hypertrophy) (Faust et al., 1979; Bjorntorp, 1979). The origin of adipocytes is still unknown. They derive from mesodermal tissue and are thought to result from the reprogramming of committed fibroblastlike cells, called preadipocytes, populating the adipose tissue (Green, 1979). The reprogramming takes place during embryonic life, but some evidence indicates that the formation of new adipocytes can also occur in adults (Faust et al., 1978). Preadipocyte cells constitute a pool of cells in the stromal-vascular fraction of the already developed adipose tissue. Bjorntorp and co-workers (1978) succeeded in cultivating cells isolated from the stromal-vascular fraction and showed that the cells could undergo proliferation and adipose conversion in culture. But the identification of the replicating preadipocyte or differentiated adipocyte has yet to be accomplished (Hausman et al., 1980).

A glycerol-ester hydrolase from rat intestinal cells has been purified using chromatography on carboxyhexanoyl-Sepharose-glyceryldioctanoate and preparative gel electrophoresis. The enzyme gives a single band by analytical gel electrophoresis; it is a monomer of molecular weight 68000. The optimum pH for its action on glyceryl tributyrate is between 8.0 and 8.5; the activation energy was calculated to be 8.7 kcal x mol-1 (36.4 kJ/mol). Its substrate specificity is mainly directed against esters of glycerol and of primary monoalcohols. Similarly to pancreatic lipase but contrary to liver esterase, it is inhibited by bile salts; relief of this inhibition by colipase is only observed for pancreatic lipase. The possible role of the glycerol-ester hydrolase in the absorption of short and of medium chain triglycerides is discussed.

Thymic endocrine epithelial cell line TEA3A1 can be maintained and passaged in a serum-free WAJC404A medium supplemented with insulin, transferrin, dexamethasone and EGF. EGF not only promotes the growth of these cells but also regulates the activation of phospholipase A2 enzyme activity. The binding of [125I]EGF to the TEA3A1 cells is temperature and time dependent, saturable and can be blocked by excess unlabelled EGF. Two classes of EGF receptors are found on these cells. One with Kd of 5 X 10(-11)M (approximately 3000 sites/cell) and the other with Kd of 5 X 10(-9)M (approximately 30,000 sites/cell). The resynthesis of EGF receptor in TEA3A1 cells after down-regulation requires about 24 hrs and can be blocked by both actinomycin D and cycloheximide.

The preferential cellular distribution in the villus tip and the subcellular localization in the endoplasmic reticulum of an intestinal glycerol-ester hydrolase from rat mucosa are described. The enzyme is shown not to be from either pancreatic or bacterial origin; it catalyzes the hydrolysis of short- and medium chain triglycerides and of p-nitrophenylacetate. Contrarily to the specificity found for the pig intestinal lipase (Serrero, Negrel and Ailhaud, 1975), no activity is detectable against acylCoA; a thiolester hydrolase different from the glycerol-ester hydrolase was demonstrated after differential solubilization and chromatographic separation. A high proportion of glycerol-ester hydrolase is present in the intestinal lumen; its possible complementary role in lipid degradation is discussed.