Fertility, Kinases, and Cancer

By Jason Socrates Bardi

"The one without born of the one within."

From Paradiso: Canto XII by Dante Alighieri.

Last week a Manchester newspaper reported that Louise Joy Brown, 24, is engaged. Though of note in this British industrial city, where Ms. Brown is something of a minor celebrity, the story was not widely reported worldwide.

Twenty-five years ago, however, the news that Brown, the world's first "test-tube" baby, had been born was widely and exhaustively reported. She was the Dolly of the decade, the Clonaid baby of the late 70s, and her birth heralded doom in the view of some and a new age in assisted reproduction for others. Today, the fact that Brown's news does not make the wires is a measure how far in vitro fertilization and fertility science has come in just one generation.

In the world of fertility science, the last 25 years have been most productive. In the year 2000, the most recent for which the U.S. Centers for Disease Control and Prevention makes data available, more than 400 fertility clinics were operating in the United States alone, and nearly one out of every hundred children born in this country was born thanks to assisted reproductive technologies like in vitro fertilization.

Yet infertility in both men and women still poses myriad problems, and some of its basic causes remain unexplained.

Now a team of scientists led by Professor Steven Reed and Research Associate Charles Spruck, both members of the Department of Molecular Biology at The Scripps Research Institute (TSRI), has identified a mammalian protein that seems to play a crucial role in "meiosis," a biological process critical for fertility.

Certain female fertility problems arise from an inability in a woman's ovum, or egg, to complete the complicated process known as oogenesis, whereby it divides and matures through sequential "mitotic" and "meiotic" divisions in order to prepare the egg for fertilization by a man's sperm. Male fertility problems can occur because the early sperm cells, called "spermatocytes," are not able to undergo the same sort of meiotic division to produce viable sperm.

It seems strange that a single protein would have such a profound biological effect on the fertility of both men and women. After all, the ovaries and the testes are two very different organs, and the process of meiosis that occurs in both spermatocytes and oocytes is different in these two types of cells.

Nevertheless, a protein called "Cks2" that Reed, Spruck, and their colleagues describe in the April 25, 2003 issue of Science seems to be critical for both male and female fertility. Knocking out the protein in vivo blocked both spermatocyte meiosis in males and oocyte meiosis in females.

"Any increase in our understanding of how meiotic divisions occur may help in addressing these fertility problems," says Reed, though also cautioning, "it's [too] early to say how."

Also, in knocking out Cks2 from mammals, the team has created in vivo models that can potentially serve for studying the process of human fertility disorders and meiosis.

Sexual Reproduction Starts With Cell Division

Meiosis is a special kind of cell division that prepares germ cells—the male sperm cells and the female eggs—for sexual reproduction. The process of meiosis is not completely understood, largely due to the difficulty of extracting germ cells to study.

What is special about meiosis is that it reduces the number of chromosomes a cell normally has by half. Germ cells are "haploid," which means that they have only one set of chromosomes (a total of 23). This allows the germ cell to fuse with another haploid germ cell to form a "zygote" in which the two sets of chromosomes from the two parents are combined (giving a total of 46 chromosomes). Thus, a haploid sperm can combine with a haploid egg and result in a fertilized embryonic cell.

Meiosis contrasts to mitosis, the normal process of cell division where one cell makes two copies of its DNA and splits into two "diploid" daughter cells, which each contain two copies of the parent cell's chromosomes.

The paper that Reed and his colleagues published in Science last week describes a knockout model of the Cks2 protein. The Cks2 knockouts are normal in every way except that they are sterile. "Without Cks2," says Reed, "the cells become hung up [in meiosis] and cannot proceed any further."

When members of the team analyzed the model's gonads, they found a severe defect in the male spermatogenesis, the process where sperm is generated in the testes. Without the Cks2 protein, the process became stalled in one particular stage of meiosis. Similarly, they found a defect in the female counterparts during oogenesis, the meiotic formation and maturation of the female ovum or egg.

"What we found is that oogenesis and spermatogenesis are blocked at the exact same stage in meiosis," says Reed.

If the sperm cell cannot divide, it dies before it has a chance to fuse with an egg. An egg cell without the Cks2 protein similarly never reaches the stage where it can be fertilized by a sperm.

"We don't really know why this happens," says Reed, "but what this [study] suggests is that the Cks2 protein is [functionally] required for some early aspect of meiosis."

In order to demonstrate the dependence of meiosis on the Cks2 protein, Reed turned to two collaborating groups, led by Peter Donovan of the Kimmel Cancer Center at Thomas Jefferson University, who specializes in sperm cell and oocyte development, and Richard Schultz of the University of Pennsylvania, who specializes in oogenesis.

The team isolated oocytes that would have been stuck in meiosis (because they were derived from Cks2 knockout females) and injected them with an RNA that produces the Cks2 protein. "That rescued them and allowed them to go through normal meiosis," says Reed.

A Cousin to the Rescue

Interestingly, the protein "Cks1" (a close cousin of Cks2) was able to rescue meiosis in the knockout oocytes as well. And both of these proteins, Cks1 and Cks2 were able to rescue a division problem in yeast cells caused by loss of the yeast Cks1 protein. The replacement was not perfect, but the cells were viable.

Mammalian and yeast Cks proteins are "homologs." Homologs are genes in different species that originated in a common ancestral gene long ago. In this case, the Cks genes in the different species have retained about 50 percent amino-acid identity and much of the same function. Mouse and human Cks1 and Cks2 are 80 percent identical.

"The [Cks family of genes] are structurally and functionally conserved through about a billion years of evolution," says Reed.

The Cks proteins were originally discovered in yeast—a useful organism to study given that both yeast and mammalian cells are eukaryotic and have many of the same basic proteins and mechanisms. Discoveries in yeast also often drive discoveries in mammalian cells.

After the first Cks protein was discovered in yeast, the two orthologs Cks1 and Cks2 were discovered in insects and mammals, and Reed has been studying them—and the original yeast Cks protein—ever since.

The observation that either Cks1 and Cks2 are able to rescue meiosis in the Cks2 knockouts is consistent with the finding that Cks1 is not present in meiosis. If it were, it would already have come to the rescue.

Why Cks1 is not there is not so clear. However, one possible reason is that Cks1 (but not Cks2) is normally involved in proteolysis, the process whereby proteins are chewed up inside the cell and recycled for their amino acids. During meiosis, it may be more important to maintain certain proteins and not turn them over, which may be why the Cks1 protein is not widely expressed at this stage.

Interestingly, a study Reed and his colleagues published about a year ago reported the results of knocking out the Cks1 protein. Mice without the Cks1 protein were fertile and appeared normal except that they were slightly smaller than normal (by 15 to 20 percent).

"They just didn't grow as well when they were developing," says Reed, "because the Cks1 protein is required for the turnover of an inhibitor of growth." Without the Cks1 protein, he explains, the growth inhibitor stays around and the organisms end up slightly smaller than they should be.

Interestingly, even though both the Cks1 knockout and the Cks2 knockout are viable, the double knockout is not. These models do not survive past early embryonic development.

"This suggests that even though [Cks1 and Cks2] have evolved enough to have separate functions, there is a redundant, shared function," says Reed.

Reed has reason to think that this shared function somehow involves the regulation of transcription.

Other Members of the Cell Cycle Family

The Cks proteins were originally discovered in yeast because of their interaction with another protein family that Reed has studied for over 15 years called the cyclin-dependent kinases. These are one of the crucial regulatory enzymes controlling cellular division and the cell cycle, a biological process that Reed has been studying for more than 30 years—since his days in graduate school.

The cyclin dependent kinases are binary proteins belonging to a large family of eukaryotic kinases—enzymes that exploit an abundant molecule in the cell known as ATP to attach phosphate groups to other proteins in the cell. In mammals, there are several different "cyclins" and a few different kinases that can come together at various points in the cell cycle to carry out specific phosphorylations.

In general, phosphorylation acts as a cellular "signal" that can do everything from turning the proteins on or off, controlling their transport, or even regulating survival of the cell.

"This is one of the primary modes of biological regulation," says Reed. And in the case of the specific action of cyclin-dependent kinases, he adds, "These phosphorylation events drive both mitosis and meiosis."

Cyclin dependent kinase-mediated phosphorylation is that event that gets the cell's transcription machinery running, duplicating the cell's DNA at just the right moment late in the cell cycle before a cell divides during mitosis. Naturally, these proteins must themselves be highly regulated, accumulating rapidly when they are needed to accomplish a specific task and disappearing when they are not. One of the primary modes of regulation is the degradation of the cyclin subunits when they are not needed.

Reed discovered one of these cyclin subunits, cyclin E, in the early 1990s and has since spent a considerable amount of time studying it. The protein activates by binding to the cyclin dependent kinase 2 protein and together the complex is involved in the initiation of DNA replication.

One facet of Reed's work is concentrated on the cyclin dependent kinases, their regulation, and their interactions with other proteins in the cell—besides those that they phosphorylate. The Cks proteins are a facet of this work. In meiosis, the cell cycle is controlled by the Cdk–Cks complex, the structure of which Reed and TSRI Professor John Tainer solved a few years ago. Reed's research suggests that the Cks proteins seem to serve as "adaptors" that allow the cyclin dependent kinases to interact more efficiently with their molecular targets.

Another facet of his work is how the cyclin dependent kinases and their regulation are relevant to human cancer. He has a multifaceted project that aims to understand protein turnover, the role of Cyclin E in carcinogenesis, and how the deregulation of the protein causes cell proliferation and cancer.

"Cancer is a disease of cell proliferation," he says. "And one of the reasons we study [the normal machinery of] cell proliferation is to understand how it works so that we can figure out what goes wrong."

CDK and Cancer

In some malignant cancer cells, the levels of cyclin E do not drop. When cyclin E is overexpressed, cells become genomically and genetically unstable—gaining and losing chromosomes. "This chromosome instability," says Reed, "is one of the hallmarks of cancer."

Reflecting the complexity of the cell, the loss of control is not necessarily related to problems with the cyclin E itself but rather problems with one protein that is supposed to control it.

Last year Reed published a study looking at another cellular protein called "hCdc4" which is a specificity factor for the cells' ubiquitin ligase machinery, which degrades proteins. The cell uses hCdc4 to target cyclin E to help the cell turn it over.

Reed discovered evidence suggesting that hCdc4 is a tumor suppressor protein—its presence works to counter the proliferation of cancer cells. He found that in certain types of cancer, particularly endometrial cancer, the hCDC4 gene is often mutated. Reed's group is now analyzing breast cancer samples for similar hCdc4 mutations. These mutations create forms of the hCdc4 protein that fail to target cyclin E, and this leads to the accumulation of cyclin E in the cell. The accumulation of cyclin E leads to the chromosomal instability, which is known to contribute to cancer.

Reed is trying to work out how and why cyclin E is turned over in order to address some of the problems that it causes with regard to cancer. Similarly, he is trying to figure out the role of the Cks proteins in all of this. Gene expression analysis studies of several different tumor cells have shown that Cks2 (and to a lesser extent Cks1) is one of the most frequently overexpressed proteins in cancer cells.

"Overexpression of this protein may be deleterious to cell integrity and [may be] part of the process of malignant transformation," says Reed, adding that he would like to know why, and upon finding out why would like to use that knowledge to formulate a strategy to stop it.

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"Any increase in our understanding of how meiotic divisions occur may help in addressing... fertility problems," says Professor Steven Reed. Photo by Biomedical Graphics.