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[in Menopause - Hormones and Cancer Proceedings]
edited by M. Neves -e-Castro and B.G. Wren 2002
[CH 4 p23-36]

Outline and Commentary on the 2002 Proceedings
by Timothy D. Bilash MD, MS, OBGYN
June 2003

J. Russo, MH Lareef, Q. Tahin, Y.F. Hu, C. Slater, I. H. Ruso [MHC CH 4]
Breast Cancer Reasearch Laboratory
Fow Chase CAncer Center
7701 Burholme Avenue
Philadelphia, PA 19111

Chromosome, Growth Studies

  1. Era (estrogen alpha) receptors
    1. reside in the nucleus in an inactive form associated with large inhibitory protein complexes
    2. all estrogens (endogenous and exogenous including DES) bind and activate the receptor
      1. conformational change and dimerization takes place, causing binding to transcription factors (AP-1) which recruits co-activators (SRC-1)
      2. also forms ternary complex with a coactivator protein after interacting with regulatory sequences in promotor genes
    3. estrogen alternative non-receptor pathways
      1. overexpression of p21 induces Er and Er response promoters, in an estrogen-responsive manner without receptor

  2. Estradiol is converted to estrone, and both to catechol estrogens by cytochrome P450
    1. catechol estrogens induce an estrogen response
    2. not thru Er receptors (not inhibited by antiestrogen)
    3. estrogen may not need to activate its nuclear receptor to initiate or promote breast carcinogenesis
      1. estrogen and estrogen metabolites, P450 intermediates may exert direct genotoxic effects/ mutation
      2. still needs to be demonstrated in normal breast epithelium
      3. elevated synthesis or monomethylation lead to semiquinones and to quinones both of which are electrophiles that may be carcinogenic reactive intermediates in peroxidase activation

  3. Human breast model (HBEC MCF-10F)
    1. MCF-10 is spontaneously immortalized cell line, lacking Era, Erb receptors
    2. Erb receptors are induced in cells transformed by chemical carcinogens
    3. treated with estradiol and DES over 2 weeks (4 applications)
    4. estradiol and DES showed some similar effects as MCF-10F cells transformed by benzapyrene (BP) or oncogenes

  4. Analysis of specific genome alterations indicative of neoplastic transformation

    1. microsatellites are polymorphic markers used to map the gene loci
      1. highly polymorphic, very common
      2. origin not well established, may result from polymerase errors
      3. used to mark allelic losses present in specific gene regions of transformed clones

    2. LOS- loss of heterozygosity >50% reduction in one of the heterozygous alleles at known pertinent loci affected in ductal hyperplasia, carcinoma in situ, invasive carcinoma
      1. BP treated cells did not exhibit LOH at any of the loci tested (chromosome 3 or 11)
      2. only clones DES-5 in chromosome 3, and clones E2-1, E2-2 in chromosome 11, exhibited LOH

    3. most frequent allelic loss found with breast cancer is in chromosome 17, not yet found with estrogen treatments

    4. deletions in 3p gene regions are not considered specific for breast cancer, but implicated as a genetic event triggered by DES
      1. estradiol and BP were NOT seen to cause LOH at chromosome 3 sites as DES did
      2. suggestion of suppressor genes in 3p regions
      3. 3p21.3 LOH found more frequently in metastatic than primary tumors
      4. 3p21.1-14.2 region deletions seen frequently with in situ carcinoma, benign tumors, familial breast cancers and is associated with dysregulated cell proliferation rather than tumor progression

    5. chromosome 11 contains LOH regions in breast, colon and other cancers, possible supressor gene dysfunction affecting early onset breast cancer and metatstatic processes
      1. significance of LOH in these regions and effects of estradiol needs to be clarified

  5. analyzed for phenomic alterations indicative of neoplastic transformation
    1. doubling time
      1. doubling time only slightly decreased with Estradiol and DES treatment (78/73 vs 93)
      2. doubling time markedly decreased >50% with BP treatment (42 vs 93)
      3. [see tab 1 p26] markers of cell transformation
    2. colony counts
      1. DES clone = 151
      2. BP clone = 89
      3. E2 clone = 24
      4. control didnt form colonies = 0
    3. ductulogenesis (a measure of more normal ductal/non-solid component of tumor growth)
      1. decreased in estradiol treated cells, as well as DES
      2. [see fig 2 pg25] ducts vs solid growth
    4. induction of anchorage-independent growth
      1. increased in estradiol and DES treated cells

  6. so still a confusing picture
    1. gene effects: were rare with estradiol- and DES- treated cells. LOH was a rare event manifested in different chromosomes and in only a few clones, and not at all in BP treated cells
    2. growth effects: phenotypic changes were induced by both estrogens (17bE2, DES) and chemical carcinogen (BP)
      1. estradiol increases proliferation (without branching)
      2. short-term treatment with 17betaEstradiol or DES induces anchorage-independent growth and colony formation in agar-methocel, and reduced duct formation in collagen gel, phenoytype expressions of neoplastic transformation which are also induced by BP under the same culture conditions.
      3. however, BP (tumorogenic) decreases proliferation, so proliferation does not necessarily indicate carcinogenesis
      4. estradiol effects are small compared to tumor inducing BP or DES

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