39 research outputs found
Regulation of globin gene expression: boxes and borders
The experimental work presented in this thesis pertains to the regulation of gene expression
and focuses on the regulation of the B globin genes. The expression of the globin genes is
restricted to the erythropoietic tissue and is regulated during development. Therefore the study
of globin gene expression can yield valuable insights in how organisms regulate their genes in
a coordinated tissue and developmental manner.
Two main issues will be addressed in the chapters of this thesis. The first concerns the
mechanism by which a transcriptionally competent domain is formed in the chromosomal
environment. This topic is the subject of Chapter 2. Once transcriptionally competent domains
are formed, gene transcription can take place and it is orchestrated by factors that interact
specifically with the regulatory elements of the gene itself. This interaction determines tissue
specificity and developmental regulation of a gene. The second issue addressed concerns the
transcriptional regulation of the ~ globin gene via their CCAAT boxes studied through NFE6,
a factor that binds to these elements (Chapters 3 and 4). NF-E6 interaction with its target
sequence participates in the change in globin gene expression concomitant with the
progressive changes that occur during the development of an embryo to a fetus
The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene
The beta-globin locus control region (LCR) is contained on a 20 kb DNA
fragment and is characterized by the presence of five DNaseI
hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active
6.5 kb version of the LCR, called the muLCR, has been described.
Expression of the beta-like globin genes is absolutely dependent on the
presence of the LCR. The developmental expression pattern of the genes in
the cluster is achieved through competition of the promoters for the
activating function of the LCR. Transgenic mice experiments suggest that
subtle changes in the transcription factor environment lead to the
successive silencing of the embryonic epsilon-globin and fetal
gamma-globin promoters, resulting in the almost exclusive transcription of
the beta-globin gene in adult erythropoiesis. In this paper, we have asked
the question whether the LCR and its individual hypersensitive sites
5'HS1-4 can activate a basic promoter in the absence of any other globin
sequences. We have employed a minimal promoter derived from the mouse
Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The
results show that the muLCR and 5'HS3 direct erythroid-specific, embryonic
expression of this construct, while 5'HS1, 5'HS2 and 5'HS4 are inactive at
any stage of development. Expression of the muLCR and 5'HS3 transgenes is
repressed during fetal stages of development. The transgenes are in an
inactive chromatin conformation and the lacZ gene is not transcribed, as
shown by in situ hybridization. These data are compatible with the
hypothesis that the LCR requires the presence of an active promoter to
adopt an open chromatin conformation and with models proposing progressive
heterochromatization during embryogenesis. The results suggest that the
presence of a beta-globin gene is required for LCR function as conditions
become more stringent during development
Coamplification of DAD-R, SOX5, and EKI1 in human testicular seminomas, with specific overexpression of DAD-R, correlates with reduced levels of apoptosis and earlier clinical manifestation
Seminomas and nonseminomas represent the invasive stages of testicular
(TGCTs) of adolescents and adults. Although TGCTs are characterized by
extra copies of the short arm of chromosome 12, the genetic basis for gain
of 12p in the pathogenesis of this cancer is not yet understood. We have
demonstrated that gain of 12p is related to invasive growth and that
amplification of specific 12p sequences, i.e., 12p11.2-p12.1, correlates
with reduced apoptosis in the tumors. Here we show that three known genes
map within the newly determined shortest region of overlap of
amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to
the telomeric region of the SROA, DAD-R is the first gene at the
centromeric region within the 12p amplicon. Although all three genes are
amplified to the same level within the SROA, expression of DAD-R is
significantly up-regulated in seminomas with the restricted 12p
amplification compared with seminomas without this amplicon. DAD-R is also
highly expressed in nonseminomas of various histologies and derived cell
lines, both lacking such amplification. This finding is of particular
interest because seminomas with the restricted 12p amplification and
nonseminomas are manifested clinically in the third decade of life and
show a low degree of apoptosis. In contrast, seminomas lacking a
restricted 12p amplification, showing significantly lower levels of DAD-R
with pronounced apoptosis, manifest clinically in the fourth decade of
life. A low level of DAD-R expression is also observed in normal
testicular parenchyma and in parenchyma containing the precursor cells of
this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression
in seminomas and nonseminomas correlates with invasive growth and a
reduced level of apoptosis associated with an earlier clinical
presentation. These data implicate DAD-R as a candidate gene responsible
in part for the pathological effects resulting from gain of 12p sequences
in TGCTs. In addition, our results also imply differences in expression
regulation of DAD-R between seminomas and nonseminomas
Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis
Human testicular germ-cell tumors of young adults (TGCTs), both seminomas
and nonseminomas, are characterized by 12p overrepresentation, mostly as
isochromosomes, of which the biological and clinical significance is still
unclear. A limited number of TGCTs has been identified with an additional
high-level amplification of a restricted region of 12p including the K-RAS
proto-oncogene. Here we show that the incidence of these restricted 12p
amplifications is approximately 8% in primary TGCTs. Within a single cell
formation of i(12p) and restricted 12p amplification is mutually
exclusive. The borders of the amplicons cluster in short regions, and the
amplicon was never found in the adjacent carcinoma in situ cells.
Seminomas with the restricted 12p amplification virtually lacked apoptosis
and the tumor cells showed prolonged in vitro survival like seminoma cells
with a mutated RAS gene. However, no differences in proliferation index
between these different groups of seminomas were found. Although patients
with a seminoma containing a homogeneous restricted 12p amplification
presented at a significantly younger age than those lacking it, the
presence of a restricted 12p amplification/RAS mutation did not predict
the stage of the disease at clinical presentation and the treatment
response of primary seminomas. In 55 primary and metastatic tumors from 44
different patients who failed cisplatinum-based chemotherapy, the
restricted 12p amplification and RAS mutations had the same incidence a
Tumour genomic and microenvironmental heterogeneity as integrated predictors for prostate cancer recurrence: a retrospective study
Clinical prognostic groupings for localised prostate cancers are imprecise, with 30–50% of patients recurring after image-guided radiotherapy or radical prostatectomy. We aimed to test combined genomic and microenvironmental indices in prostate cancer to improve risk stratification and complement clinical prognostic factors
Tumor senescence and radioresistant tumor-initiating cells (TICs):Let sleeping dogs lie!
Preclinical data from cell lines and experimental tumors support the concept that breast cancer-derived tumor-initiating cells (TICs) are relatively resistant to ionizing radiation and chemotherapy. This could be a major determinant of tumor recurrence following treatment. Increased clonogenic survival is observed in CD24(-/low)/CD44(+ )TICs derived from mammosphere cultures and is associated with (a) reduced production of reactive oxygen species, (b) attenuated activation of γH2AX and CHK2-p53 DNA damage signaling pathways, (c) reduced propensity for ionizing radiation-induced apoptosis, and (d) altered DNA double-strand or DNA single-strand break repair. However, recent data have shed further light on TIC radioresistance as irradiated TICs are resistant to tumor cell senescence following DNA damage. Taken together, the cumulative data support a model in which DNA damage signaling and repair pathways are altered in TICs and lead to an altered mode of cell death with unique consequences for long-term clonogen survival. The study of TIC senescence lays the foundation for future experiments in isogenic models designed to directly test the capacity for senescence and local control (that is, not solely local regression) and spontaneous metastases following treatment in vivo. The study also supports the targeting of tumor cell senescence pathways to increase TIC clonogen kill if the targeting also maintains the therapeutic ratio