Introduction

The onset of breast cancer in women is one of the most devastating diseases known today, afflicting approximately one in nine women in Western countries. In families that inherit breast and ovarian cancer, BRCA1 mutations account¹ for close to 100% of resultant cancers, and in pedigrees that solely inherit breast cancer, BRCA1 alterations are present in nearly two-thirds of the families.² These findings have led to the terminology of BRCA1 as a true tumor suppressor. Its discovery in 1994 initially did not lead to any insights into its functions, as the domains of the 220-kDa protein were not exceptionally homologous to any known proteins.³ However, research into the function of BRCA1 has yielded several theories regarding its purpose in normal cells. Identification of interacting proteins, production of antibodies against the protein, development of knock-out and transgenic mouse models and comparisons between BRCA1 wild-type and mutant-expressing cells has assisted in placing functional characteristics with the BRCA1 protein. Among other qualities of BRCA1, it has become clear that it is influenced by and affects directly the position of the cell cycle. From phosphorylation to subcellular localization, protein-protein interaction to transcription activation, it has become clear that BRCA1 activities are closely related with cell cycle events. The transition from phase to phase in the mammalian cell cycle intimately involves the BRCA1 tumor suppressor.

BRCA1 Protein and mRNA During the Cell Cycle

Detection of BRCA1 transcripts initially did not identify a cell cycle component to its regulation. However, a study looking specifically at the status of BRCA1 mRNA in G0 cells found that the transcript was greatly reduced.⁴ While expression was high in exponentially growing cells, withdrawal of growth factor from human mammary epithelial cells resulted in a disappearance of BRCA1 altogether. In addition, senescent cells also had dramatically reduced BRCA1 transcript. The lack of BRCA1 transcript in non-dividing cells led to the notion that this may be a component of the cell division machinery. At least in the case of senescence, recent reports of the ability of p53 to repress the transcription of BRCA1 may underlie this result.^5,6 Another contributor to the regulation of BRCA1 mRNA expression is the pRb-E2F complex.⁷ The promoter of BRCA1 contains several E2F binding sites and pRb is able to repress transcription from the BRCA1 promoter in an E2F-dependent fashion.

Development of antibodies against the full-length BRCA1 protein further established its role in the cell division process. Several papers described the shift of the BRCA1 protein band to a higher mobility upon the onset of DNA replication, or if cells had been arrested in S phase by such agents as hydroxyurea.^8–11 This shift was apparently due to phosphorylation, as addition of phosphatase to extracts resulted in a collapse of the band to its normal state, and phosphorylation occurred predominantly on serine (Figure 1). The phosphorylation of BRCA1 continued throughout S and onto the G2/M phases, after which it was progressively dephosphorylated. Blockage of cell cycle progression also resulted in phosphorylation, either at the G1/S border or at G2/M by treatment with colchicine. Later, BRCA1 was found to possess a cdk2 phosphorylation site at serine 1497.¹² This site was found to be efficiently phosphorylated in vitro by cdk2 complexed with either cyclin A or E. Kinases complexed with cyclin D have also been shown to phosphorylate BRCA1.⁹ Therefore, at least one of the kinases that force the hyperphosphorylation of BRCA1 at S phase is a major component of the cell cycle machinery. To date, there exist several other kinases that are capable of BRCA1 phosphorylation including casein kinase 2, DNA damage-responsive kinases such as ATM, ATR, hCds1 and the AKT kinase as stimulated by heregulin.^13–20 Whether these kinases also play a role in cell cycle-mediated alteration of BRCA1 remains to be tested. Also, it remains to be seen if cdk2 is capable of phosphorylating BRCA1 as late as G2/M phase—a phase that retains high levels of phosphorylated BRCA1 yet has classically been thought to possess low levels of cdk2 kinase activity. One report suggests that BRCA1 is actually predominantly tyrosine phosphorylated at G2/M stages, so there may be other cell cycle-regulated kinases that are able to affect BRCA1 phosphorylation status at different stages of the cycle.²¹ Thus far, the only concrete effect of phosphorylation on BRCA1 to be shown has been a change in protein-protein interaction with such regulators of its transcriptional activity as CtIP.²² There exists correlative evidence suggesting that phosphorylation may affect subcellular localization and the conferring of sensitivity to DNA damage,^8,16,19 but no data thus far have placed a functional significance on cell cycle-dependent phosphorylation of BRCA1.

Figure 1

The subcellular localization of BRCA1 changes with respect to the cell cycle. In G1 phase of the cell cycle, BRCA1 is typically expressed in low amounts, and what protein is present is distributed ubiquitously throughout the nucleus (Black is the cytoplasm, (more...)

Subcellular Localization

One of the first reports describing a cell cycle-dependent phosphorylation phenotype of BRCA1 also noted a peculiar cellular distribution of BRCA1 (Figure 2). In cells that were undergoing DNA synthesis, BRCA1 protein appeared as foci when immunofluorescence staining was performed.⁸ When S phase was interrupted by treatment with hydroxyurea or DNA-damaging agents, these foci dispersed, indicating a correlation of the foci with ongoing DNA replication. Proteins that are known to associate with BRCA1 such as BARD1 and Rad51 colocalized with BRCA1 to these foci. Interestingly, during S phase these foci also colocalized with PCNA-positive replication structures, suggesting that BRCA1-containing complexes are an integral part of the DNA replication machinery present at the replication fork. It may be that BRCA1 is essential for maintaining high fidelity replication in concert with its associated repair proteins such as Rad51.

Figure 2

Accumulation and phosphorylation of BRCA1 during the cell cycle. In early G1 phase, BRCA1 protein is expressed at very low levels and is at best underphosphorylated. As the cell progresses towards the DNA replication checkpoint, protein levels accumulate (more...)

At another phase of the cell cycle however, BRCA1 possesses an entirely different localization. In mitosis, the nuclear membrane breaks down, allowing the normally nuclear localized BRCA1 to distribute itself around the cell. One area of concentrated BRCA1 protein is the centrosome.²³ Immunofluorescence localizes BRCA1 to the polar ends of the cell during mitosis and is able to be immunoprecipitated with antibodies against gamma tubulin, a centrosome component. Interestingly, the gamma tubulin precipitated form of BRCA1 is hypophosphorylated. This is at odds with previous data suggesting that phosphorylation of BRCA1 continues throughout S and into G2/M. Perhaps there exist multiple populations of BRCA1 in the cell. It is of note that microtubule-destabilizing agents such as nocodazole induce a strong shift in phosphorylation of BRCA1,¹⁰ raising the possibility that this phosphorylation induction may dissociate BRCA1 from the microtubule organizing centers such as the centrosome. It will be interesting to see if BRCA1 remains associated with the polar ends of the cell after treatment with colchicine or nocodazole.

In meiotic cells, BRCA1 also has specific cellular localization. Along the chromosomes during a period when synaptonemal complexes form, BRCA1 is isolated to regions that could be undergoing recombination.²⁴ This is consistent with the induction of BRCA1 that is seen during this process. Interestingly, the DNA repair protein also colocalizes with BRCA1 during this meiotic process. Perhaps the two proteins are intimately involved in the proper exchange of genetic information between homologous chromosomes in spermatocytes or oocytes.

Activity at Cell Cycle Checkpoints

One aspect of subcellular localization of BRCA1 remains constant, regardless of cell cycle phase. It is always involved in the protection of genomic DNA. Two major cell cycle checkpoints that are required for genomic stability are DNA replication, where the lack of fidelity could result in a mutation that may be deleterious to the daughter cell, and mitosis, where the separation of sister chromatids must be performed carefully lest the distribution of unequal amounts of genetic information be passed on to each cell at division. As it turns out, through knock-out and overexpression models, BRCA1 seems to be an integral component of both of these checkpoints.

The first attempt at eliminating BRCA1 from the genomes of mice resulted in very early embryonic lethality.²⁵ The complete lack of BRCA1 in developing mice forced death at embryonic day 7–8. What was interesting about these embryos is that when checked for expression levels of p21WAF1, the amounts were staggeringly high, indicative of the activation of the G1/S checkpoint. This finding suggested that sufficient DNA damage had occurred in the absence of BRCA1 enough to activate the G1/S checkpoint. BRCA1 itself may also be involved in activating this checkpoint when it is present in cells. Overexpression of BRCA1 has been shown to cause cell cycle arrest, but this effect requires the presence of p21WAF1 or pRb, both proteins that are intimately involved in the G1/S checkpoint.^26,27 Therefore, while it is clear that the G1/S checkpoint is still intact in the absence of BRCA1, some events that are necessary to take place (or to avoid) require a functional BRCA1 protein.

After the disastrous effect deletion of BRCA1 had on embryogenesis, a few studies sought to find types of BRCA1 knock-outs that would allow development of embryos, at least until fibroblasts could be harvested. It has long been known that a truncated form of BRCA1, lacking the coding sequence from exon 11, is expressed in cells, albeit to a much lower level than full length and is mostly only expressed in developing embryos.²⁸ One study developed a transgenic mouse that only expressed this truncated form of BRCA1, called BRCA1 Δ11. Mouse embryo fibroblasts derived from these animals senesced much faster than wild-type cells and harbored a plethora of genomic abnormalities. The chromosomal alterations seemed to be the result of unequal recombination and breakage—two anomalies that predominantly occur during mitosis. Indeed, these fibroblasts treated with DNA-damaging agents failed to enact their G2/M checkpoint, and progressed into mitosis as if there was no chromosomal damage. One possible factor in the increase of chromosomal breakage in BRCA1 Δ11 MEFs was the amplification of centrosomes in mitotic cells. Pulling on chromatids in several directions as opposed to just to polar ends of the dividing cell could certainly have an effect on the state of the chromosomes after anaphase. As previously mentioned, the centrosomes are a prime location for BRCA1 during this process; perhaps the lack of full-length protein at these complexes could account for the amplification. The overall lack of G2/M checkpoint control pointed clearly at the involvement of the exon 11 region of BRCA1 as an absolutely necessary factor in qualifying cells for division. The overexpression of the C-terminus of BRCA1 in normal breast epithelial cells has also been shown to adversely affect G2/M checkpoint control.²⁹

From these data, it is then clear that cells lacking a wild-type, DNA damage-responsive BRCA1 proceed throughout the cell cycle past mitosis, regardless of the anomalies that occur during mitosis. These aberrations pile up and lead to a cell that is genetically incapable of dividing. In the case of the G1/S checkpoint, BRCA1 is apparently not required to halt the cell cycle in the case of damage; however, it is necessary for proper repair of damage that may occur during or prior to S phase. On the other hand, in human cancer, mutations in BRCA1 lead to unrestricted cell growth; therefore, these transgenic and knockout models do not necessarily reflect the true in vivo nature of cancerous BRCA1 protein. In breast cancer, BRCA1 is not entirely deleted, usually mutations result in single amino acid changes in the N-terminal to middle portions of the protein or in small truncations at the C-terminus. Such miniscule mutations may permit semi-normal function of a particular cell, yet at the same time allow genetic alterations here and there to slip by the damage sensing that is otherwise detected by BRCA1. The right (or wrong, depending on your point of reference) mutations left undetected could cut the brakes on the speed of cell growth.

Interactions with Cell Cycle Proteins

While being post-translationally modified by the cell cycle machinery and also controlling critical steps of the checkpoint pathways, it seems logical that BRCA1 would associate with proteins that are part of this process. Following the findings that BRCA1 is phosphorylated in a cell cycle-dependent manner, a number of reports detailed the interaction of BRCA1 with cyclins, cyclin-dependent kinases, E2Fs and the Rb protein. Not surprisingly, BRCA1 binds cyclin-dependent kinase 2, and interacts with it as an active kinase, as immunoprecipitation of BRCA1 co-precipitates with kinase activity.¹² The activating cyclin in this complex bound to BRCA1 appears to be cyclin A. Whether or not cyclin E, another cyclin bound to cdk2 that allows phosphorylation of BRCA1 in vitro is also bound to BRCA1 in cells remains to be seen. A slew of other cell cycle machinery proteins have also been described to bind BRCA1 including cdc2, cyclins B and D, cdks 2 and 4, and E2F4; however, the functional significance of these interactions has yet to be determined.³⁰

While BRCA1 is dependent, at least in some cell types, on the presence of pRb to arrest cell growth,²⁷ BRCA1 is also able to interact with pRb.³¹ BRCA1 was found to interact directly with pRb as well as the pRb interacting proteins RbAP48/46. This binding also led to an indirect association with histone deacetylase 1 (HDAC1), significant in that there has been much speculation as to the involvement of BRCA1 in transcriptional control. This association provides at least an indirect link to an enzymatic process that has been definitively shown to be involved in transcription. The effect s these interactions have on the progress of the cell cycle or with the activity of pRb has not been found. It will be interesting to see if the phosphorylation of pRb at the G1/S border, approximately the same temporal location as BRCA1 phosphorylation, has any effect on the interaction with BRCA1.

A few other proteins that were not originally thought to play a role in cell cycle control also appear to associate with BRCA1 in a cell cycle-specific manner. BARD1, a protein that is structurally similar to BRCA1, was one of the first proteins found to bind directly to BRCA1.³² The nuclear dot pattern that is characteristic of BRCA1 during S phase also is true for BARD1. This foci formation of BARD1 only occurs during S phase, and the dots are overlapping with BRCA1 foci, indicating that association of the two proteins may be cell cycle specific.^24,33 The overall expression of BARD1 however appears to be ubiquitous throughout the cell cycle; therefore it is likely that post-translational modifications such as phosphorylation may be a determining factor for this association. While clearly being a true binding partner to BRCA1 in vivo, the function of BARD1 and the significance of BRCA1 interaction remains elusive. The copurification of BARD1 with CstF-50, an mRNA stabilization factor, could be a lead on a possible involvement in BRCA1 transcriptional control.³⁴

The BRCA1 binding protein CtIP has been characterized as a protein that is able to inhibit the transcriptional activation of promoters such as p21WAF1 by BRCA1.^35–37 It is in a complex with BRCA1 and BARD1, but in contrast to BARD1, CtIP is in fact expressed in a cell cycle-dependent manner, roughly mirroring the expression pattern of BRCA1.³⁷ The interaction of the two proteins is therefore cell cycle specific. While inhibiting BRCA1's transcriptional activity, the association has recently been shown to be broken by phosphorylation of BRCA1 by the gamma-irradiation responsive kinase ATM.²² The removal of CtIP allows BRCA1 transcription activation to proceed. As this activation of BRCA1 is true for ionizing radiation, another study has found that the CtIP interaction is stable throughout several other DNA -damaging stimuli such as UV, Adriamycin, or hydrogen peroxide.³⁷ Whether BRCA1 transcription activity is affected by these other treatments is yet to be known.

Transcription of Cell Cycle Genes

It is well established that BRCA1 is likely involved in cell cycle checkpoint maintenance and/or DNA damage sensing. However, another faction of BRCA1 research is the link of BRCA1 to transcription of specific genes (Figure 3). Early on, BRCA1 was found by biochemical purification to be associated with the RNA polymerase holoenzyme and to induce transcription from a synthetic promoter when tethered to it.^38,39 Nevertheless, these data merely associated BRCA with general transcription, not differential specific activation or repression. Indeed, no evidence had been shown that BRCA1 possesses a promoter binding sequence nor had it been able to bind DNA directly. However, much work since then has suggested that the expression of BRCA1 forces changes in the expression patterns of certain genes that are not so coincidentally linked to processes it has been proven to be involved in such as DNA damage response and cell cycle progression. BRCA1 has been shown to be bound to such transcriptional regulators as p53, c-Myc, the estrogen receptor and p300.^40–43 Its effect on each of these protein's activity is what one would expect from a tumor suppressor—coactivation of p53, inhibition of c-Myc, etc.

Figure 3

Activation of genes by BRCA1 relevant to the cell cycle. BRCA1 has been shown to control the expression levels of four genes—p21WAF1, Gadd45, Cyclin B1, and EGR1—that have implications on checkpoint activation. The p21WAF1 and EGR1 genes (more...)

Activation of the p21WAF1 gene expression was the first in a line of BRCA1-regulated genes to be discovered.²⁶ Overexpression of BRCA1 strongly activates the p21WAF1 promoter and interestingly is not dependent on p53. Expression of BRCA1 from an exogenous promoter, as previously mentioned, is able to cause growth arrest in most cell lines. This arrest in G1 phase is dependent on the presence of p21WAF1, as expression of BRCA1 in isogenic cell lines lack p21WAF1 are able to progress into DNA replication and eventually arrest in G2 phase.⁴⁸

Two reports describing expression of BRCA1 in different cell lines identified the DNA damage response gene Gadd45 as a strong target of BRCA1 as well. In both, an induction of Gadd45 mRNA was clearly visible by Northern blot analysis after BRCA1 transcript induction.

In one case, BRCA1 induction had an apoptotic effect in the U2OS cell line by use of a tetracycline-inducible system.⁴⁴ As Gadd45 had been previously shown to activate the proapoptotic c-Jun N-terminal kinase a link was drawn between JNK and subsequent apoptosis, by coexpression of a dominant negative mutant of JNK and a concomitant decrease in apoptosis seen by BRCA1 expression. However, recent evidence has suggested that Gadd45 in fact is not involved in JNK activation and that Gadd45 is not a component of apoptosis induction in vivo.^45–47 Therefore, whether BRCA1 causes apoptosis by activating Gadd45 expression, or if in fact this is a cell type-specific effect has yet to be determined.

In another case, an adenovirus expressing BRCA1 was infected into several different cell lines with varying cell cycle changes, but no apoptosis.⁴⁸ A common theme among all the cell cycle changes in each cell type was an increase in the G2/M phase content. Interestingly, Gadd45 knock-out mice possess a defective G2/M checkpoint, and the involvement of BRCA1 in the maintenance of genomic stability at mitosis has already been established.^28,47 Therefore, it is possible that BRCA1 may in part induce the Gadd45 protein in the interest of activation of the G2/M checkpoint.

Cyclin B1 is also an apparent target of BRCA1; however, its expression levels are decreased by the exogenous expression of BRCA1.⁴⁸ Again, the links to the involvement in G2/M phase control are evident. Upon the detection of anomalies in mitosis, the first step in allowing repair to proceed is to halt progression of the cycle. The inactivation of cdc2 kinase by depletion of the activating cyclin B1 is one way to accomplish this goal. In this study, a G2/M arrest induced by BRCA1 overexpression was found to be abrogated by coexpression of exogenous cyclin B1, indicating that one way by which BRCA1 is able to arrest the cell cycle at G2/M phase is by repression of cyclin B1. It will be interesting to see the possible involvement of the histone deacetylase 1 enzyme bridged to BRCA1 by RbAP46/48 in this process.

The induction of the serum responsive, early response growth factor 1 (EGR1) has also been found to be induced by BRCA1 in array screening for BRCA1 targets.⁴⁴ EGR1 belongs to a group of proteins that are involved in the progress through G1 phase of the cell cycle following growth factor stimulation, which includes c-Myc, c-Jun and c-Fos. These other immediate early genes however are not induced by BRCA1. The induction of EGR1 by BRCA1 may be indirect, owing to its late upregulation and abrogation of the JNK pathway.

Finally, the physical link of BRCA1 to the regulatory regions of these genes that it is proposed to affect has been lacking. However, a recent finding of a protein, termed ZBRK1, has provided some insight into this necessary interaction.⁴⁹ BRCA1 binds directly to ZBRK1, which in turn binds to a consensus DNA sequence defined as GGGXXXCAGXXXTTT. Interestingly, this sequence resides in many of the genes described in recent papers to be up- or down-regulated as a result of BRCA1 expression. These include p21 (3 sites), Gadd45, Gadd153, Ki-67 and EGR1. Coexpression of ZBRK1 and BRCA1 were found to actually repress the Gadd45 promoter, contrary to what one would expect given previous findings of activation of Gadd45 expression by BRCA1. Thus far, it is hypothesized that overexpression of BRCA1 may titrate ZBRK1 away from the promoter, allowing transcription to occur. In vivo, the situations may be different, as they involve such other events as phosphorylation by a number of kinases, binding to other repressors such as CtIP and cell cycle-specific localization.

Conclusion

The links between BRCA1 and the cell cycle have been made clear with solid data published over the last few years. Advances in the field have allowed visualization of BRCA1 localization during the cell cycle, during mitosis, meiosis, and DNA replication that provide insight into how BRCA1 functions with respect to the cell cycle. Expression at both the mRNA and protein levels have also been linked to this.

Further study into this association between BRCA1 and the cell cycle will allow delineation of such issues as whether BRCA1 is a regulator or is regulated by the progression of the cell cycle, how phosphorylation affects the localization and function of BRCA1 during this progress and if transcriptional control by BRCA1 is a requirement for the normal transition from phase to phase of the mammalian cell cycle clock.

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