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Use of Transcription Factors as Agents and Targets for Drug Development

Use of Transcription Factors as Agents and Targets for Drug Development

ABSTRACT: Cells respond to external signals by either activating or inhibiting key regulatory proteins of gene expression called transcription factors (TFs). Abnormal expression of these factors plays a critical role in many human cancers. Recently, many TFs have been identified and their structure-function relationship has been characterized. Such new information has been useful in designing new chemotherapeutic drugs targeting these regulatory proteins. This review discusses the use of dominant-negative (DN) mutants of TFs as inhibitors of cell growth, as well as possible strategies for screening potential inhibitors of TF activity. [ONCOLOGY 10(10):1532-1542, 1996]

Introduction

Regulation of gene expression is central to a wide variety of
cellular functions. The fate of a cell--whether it will be a muscle
or fat cell, bone, or neuron--is determined by expression of genes
in a temporal and tissue-specific manner. The cell's response
to external stimuli, such as growth factors, hormones, infections,
injury, and even stress, depends on the regulation of gene expression.
The differential expression of genes in response to signals originating
from the cell membrane determines whether a cell divides, differentiates,
or dies.

Studies on the mechanism of gene expression have identified a
group of proteins called transcription factors (TFs) as the central
regulators of gene expression. These factors bind to specific
regions of the DNA called promoters (since they promote transcription)
and enhancers (which enhance transcription from a certain promoter).

Tfs as Regulators of Gene Expression

Transcription factors can either stimulate or inhibit transcription
of target genes by regulating the formation of messenger RNA (mRNA)
and ultimately altering the production of specific proteins. Transcription
in eukaryotic cells occurs in the nucleus and is catalyzed by
RNA polymerase II and several associated TF proteins binding to
this enzyme (also known as the core initiation complex). Basal
or general TFs (TATA-binding protein [TBP] and TBP-associated
factors, TFIIA, TFIIB, TFIIE, and TFIIF) bind to the promoter
region (usually a sequence called a TATA box, although some genes
have been found to be TATA-less) in an ordered manner to initiate
transcription (Figure 1).

There are also ubiquitous TFs that recognize and bind to specific
DNA sequences in the promoter to ensure efficient transcription.
An example of this is the SpI factor. Inducible TFs are another
group of factors that also bind to specific motifs found in target
DNA sequences. These TFs are activated at specific times in specific
cells by signals from the cell membrane, and thus, have a key
regulatory function in gene expression. Examples are the early
response genes (c-fos, c-jun, c-myc), the expression of
which is induced by mitogenic signals.

Clinicians see the result of both normal and abnormal gene regulation
in their daily routine: in clinical settings, such as wound healing;
in the development of the embryo during pregnancy; and in human
diseases, such as malignancy. Abnormal gene expression gives rise
to cellular abnormalities in diseases, such as metabolic and developmental
disorders. This abnormal gene expression can result from abnormal
TF function. For instance, a mutation in the gene coding for a
TF that plays an essential role in regulating gene expression
in the pituitary (called Pit-1) has been identified in patients
with combined pituitary hormone deficiency; this mutant gene results
in growth deficiency and mental retardation.[1]

In addition, aberrant expression of genes governing cell growth
and differentiation (called proto-oncogenes) plays a critical
role in the development of cancer. An example of this is Burkitt's
lymphoma, in which the oncogene c-myc is deregulated; this results
in rapid cell growth, leading to lymphoma.[2] Mutant forms of
steroid/thyroid hormone receptors, another class of TFs, have
also been identified as the cause of resistance to hormones, such
as the glucocorticoids.[3]

Structure and Activity of TFs

The basic functional domains of TFs consist of a transcriptional
action domain (activating or repressing), a DNA-binding domain,
a dimerization domain, and a nuclear translocation signal (Figure
2A
). There are several major classes of TFs based on these structural
motifs (see Table 1)[4]: (1) helix-loop-helix (Myo-D); (2) leucine
zipper (c-Jun); (3) zinc finger (glucocorticoid receptor); and
(4) helix-turn-helix (homeobox). A recent review on the classification
of TFs can be found in reference 5.

Helix-Loop-Helix--Members of the helix-loop-helix family
of TFs oligomerize through a helix-loop-helix motif (two alpha-helices)
and interact with DNA via an adjacent basic amino acid domain.[6]
Helix-loop-helix TFs include muscle-specific transactivators (Myo-D,
myogenin, MRF-4, and myf-5). They form heterodimers that may result
in the activation of muscle-specific genes. The proto-oncogenec-Myc
is another example of a helix-loop-helix TF.

Leucine Zipper--The leucine zipper consists of an extended
alpha-helix, which contains heptad repeats of leucines that interact
with each other in a zipper-like conformation.[7] Two proteins
containing the leucine zipper motif form dimers consisting of
identical proteins (homodimers) or different proteins (heterodimers).
Members of the c-Jun and c-Fos family belong to this class and
will be discussed in more detail below.

Zinc Finger--The zinc finger motif is formed by a small
group of conserved amino acids (cysteines and histidines) binding
a zinc ion in a finger-like structure. There are two types of
DNA-binding proteins with this structure: the classic zinc finger
proteins (TFIIIA required for transcription by RNA polymerase)
and the steroid receptors (glucocorticoid receptor, estrogen receptor,
retinoic acid receptors).[8]

Helix-Turn-Helix--The helix-turn-helix motif consists of
one alpha-helix making contact in the wide groove of the DNA while
the other helix lies at an angle across the DNA. The helix-turn
helix motif was originally identified in DNA-binding domains of
phage repressors, but related forms of this motif have also been
found in homeobox (genes that determine the identity of body structure)
DNA-binding domains (ie, Pit-1, Oct-1).

TFs as Master Translators

Growth factors (eg, epidermal growth factor, platelet-derived
growth factor) and cytokines (eg, interleukin-1 [IL-1], IL-6)
are ligands that bind to specific receptors and trigger signaling
cascades that ultimately lead to cellular proliferation, differentiation,
or transformation (Figure 3). Several secondary messengers may
be activated by such ligand-receptor combinations, such as phospholipase
C-gamma, phosphoinositol kinase, guanosine triphosphate-binding
protein ras, protein kinase C, protein kinase A, and other protein
tyrosine kinases, such as mitogen-activated protein kinase (MAPK),
extra- cellular-signal-regulated kinase (ERK), and c-Jun NH2 terminal
kinase (JNK).[9] These signaling cascades eventually lead to either
activation or inactivation of nuclear TFs. Several pathways may
activate the same TF, identifying TFs as key proteins that translate
these varied signals into biologic action(s). Thus, they are important
targets for the development of inhibitory agents for a wide variety
of cellular functions.

Activating Protein-1

To give a specific example of the broad subject of TF functions
and activities, we will focus on studies done on the mammalian
TF activating protein-1 (AP-1), which is actually a complex of
the Jun and Fos family of proteins.[10,11] Activating protein-1
complexes are formed by dimers of Jun family members (c-Jun, Jun-B,
Jun-D) or heterodimers of Jun family members with Fos family members
(c-Fos, Fos B, Fra-1, and Fra-2). As shown in Figure 4, activating
protein-1 complexes bind to a specific target DNA sequence TGAC/GTCA
(also known as the tetradecanoyl phorbolacetate (TPA) response
element, or TRE) found in the promoters of several cellular genes,
such as human collagenase, stromelysin, and IL-2, and activate
the transcription of these genes.[11]

The AP-1 complex either positively or negatively regulates transcription
of the target gene, depending on the composition of the heterodimers.[12-14]
In response to different stimuli, such as growth factor stimulation,
cellular stress, or even ultraviolet light stimulation, the expression
and activity of Jun and Fos proteins are rapidly and transiently
induced.[15-17] In addition, tumor-promoting agents, such as TPA,
which is a potent activator of protein kinase C, have also been
shown to act via the AP-1 pathway.[18,19]

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