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Biomedical subjects

G J Lindeman

Publications and source records attributed to G J Lindeman.

At least 19 recordsLinked to original sources
PubMed2006

Risk-reducing surgery, screening and chemoprevention practices of BRCA1 and BRCA2 mutation carriers: a prospective cohort study.

This study prospectively evaluated the utilization of cancer risk management strategies in a multi-institutional cohort of BRCA1 and BRCA2 mutation carriers using a self-report questionnaire. Of 142 unaffected female mutation carriers, 70 (49%) had elected to receive their mutation result. Of those who knew their mutation result, 11% underwent bilateral mastectomy (BM), 29% had bilateral oophorectomy (BO), 78% performed regular breast self-examination (BSE), and 80%, 89%, 67%, and 0% had at least annual clinical breast examination (CBE), mammography, transvaginal ultrasound (TVU), and CA125, respectively. A further 20%, 7%, 0%, 21%, and 75%, respectively, reported never having had these tests. For women who elected not to receive their mutation result, 0% underwent BM, 6% underwent BO, and 77%, 42%, 56%, 7%, and 0% had regular BSE, CBE, mammography, TVU, and CA125, respectively. Only one woman used chemoprevention outside a clinical trial. Uptake of prophylactic surgery and screening was associated with knowing one's mutation status (for all behaviors except BSE), age (for BO and CBE) and residence (for mammography). In this cohort, the minority of mutation carriers utilized risk-reducing surgery or chemoprevention and a substantial minority were not undergoing regular cancer-screening tests.

Adult↗
PubMed2005

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants.

BACKGROUND: The vast majority of BRCA1 missense sequence variants remain uncharacterized for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. OBJECTIVE: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. METHODS AND RESULTS: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. CONCLUSIONS: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Adult↗
PubMed2003

Bfk: a novel weakly proapoptotic member of the Bcl-2 protein family with a BH3 and a BH2 region.

Proteins of the Bcl-2 family are critical regulators of apoptosis. Proapoptotic members, like Bax, contain three of the four Bcl-2 homology regions (BH1-3), while BH3-only proteins, like Bim, possess only the short BH3 motif. Database searches revealed Bfk, an unusual novel member of the Bcl-2 family that contains a BH2 and BH3 region but not BH1 or BH4. Bfk is thus most closely related to Bcl-G(L). It lacks a C-terminal membrane anchor and is cytosolic. Enforced expression of Bfk weakly promoted apoptosis and antagonized Bcl-2's prosurvival function. Like Bcl-G(L), Bfk did not bind to any Bcl-2 family members, even though its BH3 motif can mediate association with prosurvival proteins. Low amounts of Bfk were found in stomach, ovary, bone marrow and spleen, but its level in the mammary gland rose markedly during pregnancy, suggesting that Bfk may play a role in mammary development.

Amino Acid Sequence↗
PubMed2001

The LIM domain gene LMO4 inhibits differentiation of mammary epithelial cells in vitro and is overexpressed in breast cancer.

LMO4 belongs to a family of LIM-only transcriptional regulators, the first two members of which are oncoproteins in acute T cell leukemia. We have explored a role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. Lmo4 was expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy. Consistent with a role in proliferation, forced expression of this gene inhibited differentiation of mammary epithelial cells. Overexpression of LMO4 mRNA was observed in 5 of 10 human breast cancer cell lines. Moreover, in situ hybridization analysis of 177 primary invasive breast carcinomas revealed overexpression of LMO4 in 56% of specimens. Immunohistochemistry confirmed overexpression in a high percentage (62%) of tumors. These studies imply a role for LMO4 in maintaining proliferation of mammary epithelium and suggest that deregulation of this gene may contribute to breast tumorigenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice.

Prolactin is essential for proliferation and differentiation of the developing mammary gland. We have explored a role for Suppressor of Cytokine Signaling 1 (SOCS1) as a modulator of the prolactin response using mice deficient in SOCS1, which were rescued from neonatal death by deletion of the Interferon gamma (IFN gamma) gene. SOCS1(-/-)/IFN gamma(-/-) mice exhibited accelerated lobuloalveolar development in the mammary gland during late pregnancy and precocious lactation. Significantly, the lactogenic defect in prolactin receptor heterozygous females could be rescued by deletion of a single SOCS1 allele. These findings establish a role for SOCS1 as a negative regulator of prolactin signaling and suggest that SOCS1 is required for the prevention of lactation prior to parturition.

Alleles↗
PubMed2001

Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes.

The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein-protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO-ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional (1)H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO-ldb1 interactions.

Adaptor Proteins, Signal Transducing↗
PubMed2001

E2F4 is exported from the nucleus in a CRM1-dependent manner.

E2F is a family of transcription factors required for normal cell cycle control and for cell cycle arrest in G1. E2F4 is the most abundant E2F protein in many cell types. In quiescent cells, it is localized to the nucleus, where it is bound to the retinoblastoma-related protein p130. During entry into the cell cycle, the protein disappears from the nucleus and appears in the cytoplasm. The mechanism by which this change occurs has, in the past, been unclear. We have found that E2F4 is actively exported from the nucleus and that leptomycin B, a specific inhibitor of nuclear export, inhibits this process. E2F4 export is mediated by two hydrophobic export sequences, mutations in either of which result in export failure. Individual export mutants of E2F4, but not a mutant with inactivation of both export signals, can be efficiently excluded from the nucleus by forced coexpression of the nuclear export receptor CRM1. Similarly, CRM1 overexpression can prevent cell cycle arrest induced by the cyclin kinase inhibitor p16(INK4a), an E2F4-dependent process. Taken together, these data suggest that nuclear export contributes to the regulation of E2F4 function, including its ability to regulate exit from G1 in association with a suitable pocket protein.

Amino Acid Sequence↗
PubMed2000

The proapoptotic BH3-only protein bim is expressed in hematopoietic, epithelial, neuronal, and germ cells.

Proapoptotic Bcl-2 family members activate cell death by neutralizing their anti-apoptotic relatives, which in turn maintain cell viability by regulating the activation of the cell death effectors, the caspases. Bim belongs to a distinct subgroup of proapoptotic proteins that only resemble other Bcl-2 family members within the short BH3 domain. Gene targeting experiments in mice have shown that Bim is essential for the execution of some but not all apoptotic stimuli, for hematopoietic cell homeostasis, and as a barrier against autoimmunity. There are three Bim isoforms, Bim(S), Bim(L), and Bim(EL), which have different proapoptotic potencies due at least in part to differences in interaction with the dynein motor complex. The expression pattern of Bim was investigated by immunohistochemical staining, immunoprecipitation followed by Western blotting, and in situ hybridization. Bim was found in hematopoietic, epithelial, neuronal, and germ cells. Bim(L) and Bim(EL) were coexpressed at similar levels in many cell types, but Bim(S) was not detected. Microscopic examination revealed a punctate pattern of Bim(L) and Bim(EL) immunostaining, indicating association with cytoplasmic structures. These results are discussed in the context of the phenotype of Bim-deficient mice and the post-translational regulation of Bim's pro-apoptotic activity.

3T3 Cells↗
PubMed2000

E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control.

E2F transcription factors are major regulators of cell proliferation. The diversity of the E2F family suggests that individual members perform distinct functions in cell cycle control. E2F4 and E2F5 constitute a defined subset of the family. Until now, there has been little understanding of their individual biochemical and biological functions. Here, we report that simultaneous inactivation of E2F4 and E2F5 in mice results in neonatal lethality, suggesting that they perform overlapping functions during mouse development. Embryonic fibroblasts isolated from these mice proliferated normally and reentered from Go with normal kinetics compared to wild-type cells. However, they failed to arrest in G1 in response to p16INK4a. Thus, E2F4 and E2F5 are dispensable for cell cycle progression but necessary for pocket protein-mediated G1 arrest of cycling cells.

Alleles↗
PubMed1998

A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting.

Homozygous E2F-5 knockout embryos and mice have been generated. Although embryonic development appeared normal, newborn mice developed nonobstructive hydrocephalus, suggesting excessive cerebrospinal fluid (CSF) production. Although the CSF-producing choroid plexus displayed normal cellular organization, it contained abundant electron-lucent epithelial cells, consistent with excessive CSF secretory activity. Moreover, E2F-5 CNS expression in normal animals was largely confined to the choroid plexus. Cell cycle kinetics were not perturbed in homozygous knockout embryo fibroblasts. Thus, E2F-5 is not essential for cell proliferation. Rather, it affects the secretory behavior of a differentiated neural tissue.

Alleles↗
PubMed1997

The subcellular localization of E2F-4 is cell-cycle dependent.

The E2F family of transcription factors plays a crucial role in cell cycle progression. E2F activity is tightly regulated by a number of mechanisms, which include the timely synthesis and degradation of E2F, interaction with retinoblastoma protein family members ("pocket proteins"), association with DP heterodimeric partner proteins, and phosphorylation of the E2F/DP complex. Here we report that another mechanism, subcellular localization, is important for the regulation of E2F activity. Unlike E2F-1, -2, or -3, which are constitutively nuclear, ectopic E2F-4 and -5 were predominantly cytoplasmic. Cotransfection of expression vectors encoding p107, p130, or DP-2, but not DP-1, resulted in the nuclear localization of E2F-4 and -5. Moreover, the transcriptional activity of E2F-4 was markedly enhanced when it was invariably nuclear. Conversely, it was reduced when the protein was excluded from the nucleus, implying that E2F-4 transcription function depends upon its cytological location. In keeping with this, the nuclear/cytoplasmic ratios of endogenous E2F-4 changed as cells exited G0, with high ratios in G0 and early G1 and a progressive increase in cytoplasmic E2F-4 as cells approached S phase. Thus, the subcellular location of E2F-4 is regulated in a cell cycle-dependent manner, providing another potential mechanism for its functional regulation.

3T3 Cells↗
PubMed1995

Analysis of hematopoiesis in max 41 transgenic mice that exhibit sustained elevations of blood granulocytes and monocytes.

An unusual mouse line (max 41) that carries an inserted transgene encoding the nuclear transcription factor, Max, exhibits a 50- to 60-fold elevation of blood neutrophils and a 10-fold elevation of blood monocytes. Analysis showed that these elevated levels of blood cells were sustained incrementally by a sevenfold increase in mature neutrophils in the bone marrow and spleen and a fourfold increase in granulocyte-committed progenitor cells with normal turnover times for mature neutrophils and monocytes in the marrow and blood. the progenitor cells were not autonomous and exhibited a normal quantitative responsiveness to stimulation in vitro by the four colony-stimulating factors. A consistent anomaly noted was that, when stimulated by macrophage colon-stimulating factor, max 41 progenitor cells formed mainly granulocyte-containing colonies, rather than the usual macrophage colonies. The blast colony-forming cells from max 41 mice did not generate abnormal numbers or types of progenitor cells. The transgenic max 41 model requires further analysis to establish the regulatory anomaly responsible for the excessive production of granulocytes and monocytes, but has emphasized that most neutrophils generated in the marrow normally never leave the organ.

Animals↗
PubMed1995

Overexpressed max is not oncogenic and attenuates myc-induced lymphoproliferation and lymphomagenesis in transgenic mice.

The cellular growth promoting function of the Myc oncoprotein requires its heterodimerization with the Max protein, but Max can also form complexes that inhibit Myc action. To determine whether max overexpression in vivo is oncogenic and whether it can modulate the action of Myc, we generated transgenic mice in which the max gene was directed to express in lymphoid cells by a linked immunoglobulin heavy chain enhancer (E mu). Expression of the transgene at substantially higher levels than the endogenous max gene did not perturb lymphoid homeostasis in adult animals nor predispose to lymphomagenesis. The numbers of B-lymphoid cells in very young animals were reduced. Moreover, analysis of bi-transgenic E mu-myc/E mu-max mice revealed that max overexpression attenuated the premalignant B-lymphoproliferative state induced by an E mu-myc transgene and reduced the rate of lymphoma onset. These results suggest that elevation of Max expression in vivo inhibits the function of Myc.

Animals↗
PubMed1994

Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.

Cyclin D1 is the regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle. Deregulated cyclin D1 expression has been implicated in several human neoplasms, most consistently in centrocytic B lymphoma, where the cyclin D1 gene usually has been translocated to an immunoglobulin locus. To determine directly whether constitutive cyclin D1 expression is lymphomagenic, transgenic mice were generated having the cyclin D1 gene linked to an immunoglobulin enhancer. Despite abundant transgene expression, their lymphocytes were normal in cell cycle activity, size and mitogen responsiveness, but young transgenic animals contained fewer mature B- and T-cells. Although spontaneous tumours were infrequent, lymphomagenesis was much more rapid in mice that co-expressed the cyclin D1 transgene and a myc transgene than in mice expressing either transgene alone. Moreover, the spontaneous lymphomas of myc transgenic animals often ectopically expressed the endogenous cyclin D1 gene. These findings indicate that this G1 cyclin can modulate differentiation and collaborate with myc-like genes in oncogenesis.

Animals↗