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Our objective is to test the hypothesis that inhibition of mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 in human luteinized granulosa cells will block epidermal growth (EGF)-stimulated MAP kinase activity and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were cultured and treated with the following: (1) vehicle; (2) PD98059; (3) EGF; (4) PD98059 + EGF. Treatment with PD98059 suppressed MAP kinase activity, inhibited MAP kinase phosphorylation by Western blot analysis, blocked nuclear translocation of phosphorylated MAP kinase by confocal microscopy, and increased percentages of subdiploid apoptotic nuclei by flow cytometry. Our data are the first evidence that a relationship may exist between the MAP kinase pathway and control of apoptosis in human luteinized granulosa cells. These results support the hypothesis that suppression of the MAP kinase pathway may lead to apoptosis in these cells., (Copyright 1999 Academic Press.)

To elucidate the possible roles of proto-oncogenes and growth factors in estrogen-regulated cell proliferation of human breast and gynecologic cancers, we have determined the gene expressions of c-myc, transforming growth factor-alpha and beta 1 (TGF-alpha, beta 1) and epidermal growth factor receptor (EGFR) in a number of these cancer cell lines by using an intron-Differential (ID) RNA/PCR method, which differentially identifies the amplified cDNA from PCR products of genomic DNA contaminants. With this method, we demonstrated the expression of these genes, except EGFR, in an estrogen-dependent breast cancer cell line (CAMA-1). Our results show that TGF-alpha/EGF does not function as an autocrine factor in this cell line. Accordingly, it is unlikely that the TGF-alpha/EGFR system participates as a mediator in the estrogen-induced cell proliferation of CAMA-1 cells. The ID RNA/PCR method is a rapid, sensitive and specific technique for mRNA phenotyping and will have great clinical utility.

Studies on the mechanism of activation of estrogen receptor (ER) have led us to identify two classes of receptors. Effect of molybdate on the activation of ER was determined by their affinity for nuclei or DNA-cellulose. Molybdate caused inhibition of nuclear binding with rat uterine ER, but an increase in the binding was observed with rabbit uterine ER. Similar results were observed when DNA-cellulose was used instead of nuclei. To further test the diversity in ER activation, DNA-cellulose binding of ER from 10 primary breast cancer tissues was determined in the presence or absence of molybdate. ER from 8 tissues showed inhibition of binding, one showed an increase, and one was not affected by molybdate. These results indicate the presence of two classes of ER, one whose activation is inhibited by molybdate and another whose activation is either unaffected or stimulated by molybdate. Other differences between rat and rabbit uterine ER are also discussed.

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.