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Analogues of vitamins A and D are potent anticancer agents.

Naturally occurring retinoids (analogues of vitamin A) and vitamin D have long been known to play essential roles in normal growth and developmental processes. All-trans-retinoic acid (ATRA), and 1,25 dihydroxyvitamin [D.sub.3] (1,25 [D.sub.3]) are the most well recognized examples of these two classes of hormone-acting nutrients. Both ATRA and 1,25 [D.sub.3] act, at least partially, through specific binding proteins. Complexes of the hormone bound to receptors bind to specific sequences of DNA and modify the types and amounts of proteins produced from particular genes. Specific receptors, which are primarily located in the nucleus of the cell, have been identified for both ATRA and 1,25 [D.sub.3] and are expressed in several normal cell types and in multiple types of cancer cells. Changes in the expression of specific proteins by these hormone-complexes can profoundly alter the behaviour of the cell. ATRA has been shown to induce cellular differentiation (maturation), a distinctly different role than for retinol, which is involved in the visual cycle. As well as its documented roles in bone metabolism and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]-balance, 1,25 [D.sub.3] has been shown to induce differentiation of a variety of cancer and normal cell types.

Vitamin D deficiency most often leads to abnormal bone development; however, a more recently described deficiency symptom, is impaired bone-marrow derived macrophage differentiation. Macrophages form a critical part of the immune system, necessary for fighting off infection and also killing off precancerous or cancer cells before they have a chance to take over. They mature partly in the bone marrow, form monocytes to enter the circulating blood and then can enter tissues to mature into the end-stage macrophage. The neutrophil is a sibling of the macrophage and also performs critical roles in the immune system. The mother cell that leads to both the macrophage and neutrophil is called a "bipotent precursor" since it has the potential to form either cell type depending on the specific circumstances it finds itself in.

Leukemia results when a blood cell in the bone marrow fails to mature appropriately. It finds itself locked at a position in the developmental pathway where it continues to grow and divide, but is unable to produce the functional end cells so critical for normal tissue maintenance (the macrophage is a great garbage collector and recycling centre) and immune function. The overabundance of these immature cells prevents this cell's progeny from developing but additionally suppresses the bone marrow's ability to produce these and other mature blood cell types.

There are many different kinds of leukemia. Despite their many unique characteristics, different leukemias often respond to similar treatment strategies. Acute promyelocytic leukemia (APL) represents about 10% of acute myeloid leukemia cases and affects children and adults with a median age of 30-35. The leukemia cell in this disease has a very unique change in the genetic material which results in the exchange of information between small sections of chromosomes 15 and 17. This "translocation" causes a rearrangement of the gene that codes for one of the receptors for retinoic acid.

Leukemias are most often treated with a variety of protocols that involve the use of cytotoxic drugs and/or radiation. Chemotherapy is directed at killing the rapidly dividing leukemia cells in an attempt to clear the disease. However, one of the major consequences of such treatment is that many normal cells, which are also rapidly dividing, are simultaneously destroyed. This leads to the hair loss and gastrointestinal difficulties that so often accompany aggressive cancer treatment. An alternative strategy, and one that is gaining acceptance in the research community, is that of "differentiation therapy". In this model, instead of trying to kill the leukemic cells, one attempts to use agents that encourage the cells to mature along one of the lineages that has become blocked on the road to cancer development.

When our research group entered the arena, retinoic acid was already being used clinically to treat some patients with APL. Leukemic APL blasts require higher than normal levels of retinoic acid (about 100-1000 times normal) to differentiate into neutrophil-like cells (see Figure 1). Once the chromosomal translocation was discovered which disrupted the retinoic acid receptor, this explained the blunted response to ATRA. Most patients respond favourably to retinoic acid therapy; however, they soon relapse due to the development of retinoic acid resistance. Our bodies, and the leukemia cells themselves, are incredibly adept at responding to these pharmacologic levels of ATRA and begin producing excess binding proteins which effectively sop up the ATRA and prevent it from having is differentiating activity. This allows the few remaining leukemia blasts to repopulate and begins the disease process all over again.


Recently a cell line was developed from a patient with APL. The NB4 cell line contains the diagnostic translocation, t(15;17), and was shown to differentiate into mature neutrophils when treated with ATRA. Based on the developmental stage at which this cell type was thought to be arrested, PhD student Mickie Bhatia and I reasoned that these cells should also be capable of monocyte/macrophage differentiation. The advantage of this other pathway, we argued, would be that it may be possible to stimulate macrophage differentiation even in cells that were resistant to ATRA (i.e. leukemias that had come out of remission from ATRA therapy). Also, we hypothesized that if both pathways were stimulated simultaneously, the likelihood of eliminating all of the leukemia blasts might be higher.

Last fall, we published an article in the journal Leukemia documenting that indeed NB4 cells could be stimulated to differentiate into monocyte/macrophages by combinations of phorbol esters, 1,25 dihydroxyvitamin [D.sub.3], and macrophage colony stimulating factor (MCSF). In subsequent experiments, we demonstrated that 1,25 [D.sub.3] treatment needed to precede phorbol ester treatment for differentiation to take place and that the response was both time and dose dependent (Experimental Cell Research, in press). A substantial differentiation response could be achieved with only several minutes of 1,25 [D.sub.3] treatment. This observation coupled with the fact that the effects of 1,25 [D.sub.3] were almost completely reversed if phorbol ester treatment was delayed, suggested to us that the mechanism of action of 1,25 [D.sub.3] was not by the classical pathway.

We proposed that 1,25 [D.sub.3] acted independently of binding to the nuclear vitamin D receptor (nVDR). Instead we assumed that 1,25 [D.sub.3] was acting through a signalling pathway similar to those for growth factors. The signal would be initiated at the cell membrane, transduced through the cytoplasm and into the nucleus. In any or all of these locations, the biochemistry of the cell could be altered to affect the behaviour of the cell. By using specific chemical inhibitors of proteins previously shown to be involved in other growth factor signalling pathways, we demonstrated that the differentiating activity of 1,25 [D.sub.3] could be modulated (Exp. Cell. Res., in press). This was good evidence that 1,25 [D.sub.3] had the potential to alter cellular biochemistry outside the nucleus but did not prove that the effects were independent of nVDR.

To do this a generous colleague, Anthony Norman (University of California, Riverside) provided us with 1,25 [D.sub.3] analogues that targeted the non-genomic activities of 1,25 [D.sub.3]. These analogues (Figure 2) were locked into specific stereochemical positions that almost completely blocked their ability to bind nVDR. One of these called 6-cis 1,25 dihydroxyvitamin [D.sub.3] displays the non-genomic activities of the 1[Alpha],25 dihydroxyvitamin [D.sub.3] including modulating calcium movement across the plasma membrane. The other, 1[Beta],25 dihydroxyvitamin [D.sub.3] antagonizes the non-genomic activity of 1[Alpha],25 dihydroxyvitamin [D.sub.3].


We demonstrated that not only did the 6-cis analogue prime NB4 cells for differentiation, but it was 20-fold more potent that the authentic 1,25 [D.sub.3] which predominantly exists in the trans conformation (Journal of Biological Chemistry 1995). Furthermore, the activity of both 1[Alpha],25 dihydroxyvitamin [D.sub.3] and the 6-cis isomer were almost completely wiped out by including the 1[Beta],25 dihydroxyvitamin [D.sub.3] analogue in the differentiation mix. This represents the first example where 1,25 [D.sub.3] acting through non-genomic pathways, results in cellular differentiation. In other cell culture systems 1,25 [D.sub.3] induces an increase in expression of its own receptor, nVDR. This did not occur in NB4 cells and casts doubt on the assertion by some researchers that nVDR upregulation is essential for a differentiation response.

We are now going on to further define the molecular pathway through which 1,25 [D.sub.3] is signalling in these APL cells. We have identified one signalling molecule, called protein kinase C, as a likely second messenger for 1,25 [D.sub.3]. We are also in the process of identifying what appears to be a novel protein which responds to 1,25 [D.sub.3] by changing its state of phosphorylation on specific amino acid residues within a few minutes of 1,25 [D.sub.3] addition.

Along the lines of pursuing combination strategies that might prove to be clinically useful, PhD student Donna Berry has recently been studying the interactions between ATRA and 1,25 [D.sub.3]. We initially proposed that combinations of these agents might be more effective at inducing neutrophilic or monocytic differentiation than either agent alone. However, what we have discovered is that instead of promoting cell differentiation, these agents in combination induce a type of programmed cell death called "apoptosis". Berry is currently carrying out experiments to determine whether the nongenomic analogues of 1,25 [D.sub.3] act similarly with ATRA and is characterizing the pathways and proteins that are involved in the apoptotic process.

The pharmaceutical industry and many researchers have been directing tremendous resources and energy into developing 1,25 [D.sub.3] analogues which are more potent and have fewer side effects than naturally occurring 1,25 [D.sub.3]. Most of these initiatives have assumed that nVDR binding was essential for activity. Our data suggest that this may not always be the case and that 1,25 [D.sub.3] analogues with non-genomic activities should also be considered, not only for leukemia treatment, but for other diseases where 1,25 [D.sub.3] has shown some utility. These include osteoporosis, psoriasis and many other cancers that appear, at least in the laboratory, to respond to differentiation type therapies.

Our understanding of cancer development and treatment continues to grow at a very rapid rate. Advances continue to be made as we improve our basic understanding of the molecular mechanisms responsible for cancer evolution. By contrasting these abnormal patterns of growth with those of normal developmental processes we will then be able to design more effective intervention strategies to thwart cancer development and decrease the deleterious side effects of treatments for cancers we are unable to prevent.
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Author:Meckling-Gill, Kelly
Publication:Canadian Chemical News
Date:Nov 1, 1995
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