Cardiac myocytes begin to withdraw from the cell cycle in the second half of gestation, continue to decline through late gestation, and cease proliferating within weeks of birth. This phenomenon of cell cycle withdrawal has been studied extensively in rodents, where data shows that in the first two weeks after birth the percent of myocytes with detectable DNA synthesis drops to zero. Thereafter, cardiac myocytes demonstrate no innate capacity for regeneration.

Can a myocyte can be engineered that will both function and proliferate? Work in Bernstein's laboratory is testing their hypothesis that myocytes can be manipulated to accomplish this. Existing descriptions of myocyte-derived cell lines that proliferate while maintaining a cardiac phenotype suggest that cell division and myocyte functions are not necessarily exclusive. The feasibility of creating functioning, multiplying myocytes is based on several observations:

• cardiac myocytes in the fetus are capable of some differentiative function while maintaining the ability to proliferate
• a cell line, derived the from the atrial myocytes, has recently been described that continues to proliferate in culture while maintaining many of the features of cardiac myocytes
• micrographic figures of myocytic mitosis in patients with end-stage heart failure suggest that some mechanism is present that allows a few cells to return to division.

Although most myocytes remain quiescent from birth on, a mechanism must exist to activate their proliferation. The work in Bernstein's laboratory addresses the series of events that comprise the biology of myocytes, especially as it relates to their transcription and regulation. The group is exploring methods to manipulate this division cycle.

Ordinary cells duplicate in the S phase and separate in mitosis. This cell division requires specific, appropriate conditions for genetic material to be duplicated completely and without error, and for the mitotic spindle to be appropriately assembled. The primary oscillator driving the cell cycle is the cyclin dependent kinase (CDK) complex (Fig. 1). Cell cycle transcription and regulation is dependent on various cell stimuli and is regulated by specific CDK inhibitors. Transcription is activated by CDK4 and CDK6, which turn on the early response genes essential for cell-cycle progression. In the myocyte, the transcription activator MyoD upregulates the CDK inhibitor p21, which causes the inhibition of CDK4 and cyclin D complex (CLND), thus causing the cell cycle withdrawal necessary for myocyte differentiation.

Animals that overexpress the cyclin D complex have an increased number of myocytes. Recent research has sought to bypass the CDK control. Cultured myocytes overexpressing E2F reentered the cell cycle but then died. Transcriptional reprogramming with E2F bypasses the mitogenic requirement for cell cycle re-entry, preventing the apoptotic process. E1B protects against apoptosis by an unknown mechanism, but cells are then arrested in G2.

Bernstein's group has proposed that similar transcriptional reprogramming might bypass requirements for G2/M transit in myocytes. Cardiac myocytes re-enter the cell cycle and synthesize DNA with overexpression of G1/S-specific transcription factors. These cells remain blocked in G2, however, and failed to enter mitosis. These cells get stuck in G2, but G2/M transit requires the coordinated expression of many genes, and little is known about their transcriptional regulation. Figure 2 summarizes the current thinking about G2/M regulation. Transcriptional reprogramming may bypass requirements for G2/M.

Identification of G2/M regulators would significantly advance the understanding of this portion of the cell cycle, and provide new reagents for manipulating myocytes. Several years ago, Bernstein's group described their cloning and analysis of the human protein Cdc5 (hCdc5) in mammalian cells. This protein was remarkable because of its:

• homology to cell cycle regulators in yeast
• evolutionary conservation
• putative DNA binding domain
• existence in the cytoplasm of quiescent cells, followed by its nuclear translocation and phosphorylation with cell cycle re-entry.

Their work has also shown that overexpression of hCdc5 alters cell cycle distribution in asynchronous cultures; overexpressing hCdc5 can accelerate G2/M transit and S phase. Cells in which hCdc5 was inducibly overexpressed spend approximately the same amount of time as controls in the G1 phase, but less in S phase and significantly less time in G2/M transit.

To corroborate this finding, they created dominant negative mutant hCDc5 that contained only the amino terminal DNA binding domain. G2/M was substantially delayed in the presence of the mutant, but overexpressing wild-type hCdc5 competed against this effect (Fig. 3). This provided further evidence that hCdc5-like proteins are positive regulators of mitotic entry. Thus, overexpression of hCdc5 shortened G2, while a dominant negative mutant lacking its activation domain delayed mitotic entry_implicating hCdc5 as the first defined transcriptional regulator of G2/M in mammalian cells.

Elucidating the mechanism by which hCdc5 regulates cell division has been the focus of studies by Bernstein's group. In response to proliferative growth factors, hCDc5 undergoes nuclear transformation and phosphorylation, including additional phosphorylation in the nucleus that regulates hCDc5's interaction with downstream genes. hCdc5 appears to contain a domain capable of activation transcription, which Bernstein's group has sought to identify.

A preferential DNA binding site for hCdc5 was identified in their experiment looking for support for hCdc5's role as a binding protein. By performing cyclic amplification and selection for targets for a pool of random oligonucleotides, they identified a 12 base-pair sequence that binds to hCdc5 with affinity and specificity. The results showed that the interaction between hCdc5 and the DNA is specific, and that hCdc5 and the DNA had an affinity to the degree of other helix DNA binding proteins.

To determine if the specific binding site is present in the human genome, a yeast-based screening strategy was devised. They identified 12 clones that appeared to have the sequence that can bind to hCdc5, using a library of genomic DNA fragments and incorporating a series of counter screens. Several of these clones contained a sequence of base pairs very similar to the sequence they had previously identified. Presumably this sequence represents the regulatory elements of downstream genes for hCdc5. They also showed that the uncharacterized C-terminal of hCdc5 might contain negative regulating elements.

Thus, a high-affinity consensus sequence that binds hCdc5 through its HTH domain has been identified by Bernstein's group. They also showed this interaction occurs in vivo. Although they have searched the human genome database for a corresponding sequence in genes already characterized, this has proven difficult because this sequence presents in very small numbers in the human genome. In summary:

• Cdc5 is a positive regulator of G2/M in mammalian cells
• Cdc5 is phosphorylated and translocates to the nucleus in serum-stimulated cells
• Cdc5 binds specifically and with high affinity to a consensus, double-stranded DNA sequence
• Consensus binding sites for Cdc5 are present in low numbers in the human genome.

Defining mechanisms through which hCdc5 regulates mitotic entry may facilitate its use in reagents for manipulating the cell cycle in nondividing cardiac myocytes. Cdc5 is likely regulated through mitogen-activated signaling pathways. Phosphorylation mutants may provide reagents to manipulate the mammalian cell cycle. Cdc5 can act as a site-specific DNA binding protein. Cdc5's function in G2/M is likely mediated through DNA-protein interactions. Cdc5 targets are present in the human genome. Their identification will delineate the pathways by which Cdc5 regulates mitotic entry.