Laurens N. Ruben
Wm. R. Kenan Jr. Professor, Emeritus
Work Address: Room 138 Department of Biology, Reed College, Portland, OR 97202-8199
Home Address: 3108 SE Crytstal Springs Blvd., Portland, OR. 97202
Telephone: Work=(503) 777-7276; Home=(503) 771-6948; Fax: (503) 777-7773;
|Univ. of Michigan
||A.B.||Ann Arbor, MI||1949||Zoology|
|Univ. of Michigan
||M.Sc.||Ann Arbor, MI||1950||Zoology|
||Pre-Doctoral Fellow||NYC, NY||NCI-C-4167|
|Princeton University||Post-Doctoral Fellow||Princeton, NJ||1955||NCI-C-4167C|
The three areas of inquiry that have engaged my interest may be described as
(1) As a beginning graduate student, my attention had been drawn to observations that suggested that cancer cells shared some important characteristics with embryonic cells. If cancer cells were like embryonic cells in some ways, perhaps they would also be susceptible to some of the controls which normally guide developing cells. This might be a way of providing direction to investigators seeking to impose regulatory influences on what appeared to be an unregulated biological phenomenon, cancer. In the search for a naturally occurring developmental system on an adult vertebrate, I settled on using the limbs of certain Amphibia, in particular the salamanders, because of their ability to regenerate structurally complete and fully functional limbs after amputation. I implanted fragments from an amphibian kidney cancer and later, cells from a cancer of white blood cells, into regenerating limbs of both adults and larvae, only to find that they proved to be refractory to the developmental controls available to normal cells in their immediate environment. Much of my work at that time involved finding ways to amplify and extend the developmental processes of the regenerating limb itself. While I learned some interesting things about the limb regeneration system itself, the most exciting, consistent finding of my early experiments was that cancer cells had the capacity to induce the growth and formation of additional limb structures, e.g. digits and extra internal arm bones, a facility that comparable normal tissues did not have, unless, interestingly, their cells were in the process of dying.
(2) The next area of exploration then, grew out of these findings and led to studies which explored the growth-initiating properties of different normal tissues, as well as cancer. My hope was to identify some common attribute(s) of tissues which were particularly successful in stimulating growth. We found that this potential to initiate growth correlated well with the quantity of certain enzymes which are normally found within cells and which are usually involved in intracellular digestion of proteins. The cancerous cells leaked these enzymes into their environment, thereby dissociating neighboring tissue into individual cells which could grow, once they had been freed from their normally confining architecture. Dying normal cells were also able to leak the enzymes which were usually retained by healthy normal cells. In addition to these enzymes freeing cells from their tissue architecture, their growth stimulating capacity might have depended on the local nutritional pool of amino acids and other breakdown products supplied by the activities of these same catabolic enzymes, e.g. acid phosphatase and the cathepsins. This suggested an interesting difference between cancerous and normal cells of the same kind of tissue with respect to their capacity to transport enzymes to their external environment. This difference in the capacity to leak digestive enzymes into their local environment would seem to be of interest in thinking about how cancer cells develop the capacity to invade adjacent normal tissues.
(3) In studies with the white blood cell generated cancer (a lymphosarcoma),
I had noticed that animals which developed this cancer were unable to reject
foreign implants of tissue, which they were normally able to destroy. Since
the cancer appeared to impair an immunologic response, and little was known
then about immune reactivity in these organisms, I decided, to use amphibian
models to answer mainstream immunologic questions, which were not being successfully
addressed by using mammals. In the past, studies on the regulation of immune
responses in the "lower" vertebrates had been largely ignored. Thus,
a good deal of ground work had to be done in order to establish the information
base required before I could ask meaningful causal questions of the system.
One of the frequent questions in cancer biology has been; why is it that our bodies don`t reject cancer cells as they are produced, since they frequently are found to be different from normal cells in ways that suggest that they should be recognized and removed by our immune system. While we normally think of our immune system as functioning to protect us from invasion by foreign pathogens, e.g. bacteria, fungi and viruses, one of its principal functions is to maintain the integrity of "self" by being tolerant or unresponsive to the proteins or cells which comprise self. Thus, when cells or large molecules that can be recognized to be non-self invade our bodies, they are normally removed by cytotoxic thymus-derived (T) cells. Questions that relate to our failure to eliminate non-self on occasion, are raised about pregnancy in mammals, since the developing baby is also non-self and should be susceptible to removal by the mother`s immune system. Two situations that suggest that there may be times when the immune system is selectively compromised, systemically or locally, relate to the generation of cancer which may also display non-self epitopes (foreign domains on antigens) on its cells, and to the development of the fetus. On the basis of our own studies, we have suggested that cancer susceptibility in mammals may have evolved as the flip side of processes which led to refined capacities to distinguish self from "altered-self". Distinction of altered-self is required when most of the proteins on the cells to be monitored by the immune surveillance mechanisms are self, but some new cellular or viral gene products may now be present as a consequence of infection and or cellular transformation. Our plan of attack was to study the effects of inserting a known foreign epitope, trinitrophenyl- (TNP-), on self cells and proteins during the period of metamorphosis, when anuran amphibians (as described below) are in the process of redefining self. This protocol in Xenopus adults normally leads to a temporary tolerance, usually around 10 days, to the added foreign molecule, through the generation of molecule specific thymus-derived suppressor cells. We have explored the role of molecule-specific suppressor function in the establishment and/or maintenance of self-tolerance in Xenopus laevis, the South African clawed toad, as well as its relationship to the induction of cancer formation. We have found that during the late stages of metamorphosis the animals are most susceptible to tolerance induction to altered-self antigens. This is the very time when new adult antigens are being introduced into the metamorphosing larva. Additionally, we have studied the necessary conditions for breaking unresponsiveness to altered-self, so that an organism might be caused to destroy its own cancer. Indeed, the introduction of any combination of cytokines and antigens or lectins that will stimulate immune reactivity will lead the metamorphosing larvae to self-destruct immunologically. The same protocols may be effective locally at stimulating a patient with cancer to destroy his or her own cancer cells when they carry modified-self antigens.
Additionally, we have been studying the effects of cancer promoting reagents, e.g. the phorbol diesters, to learn their effects on a variety of immune functions. We wish to see whether the resistance of Xenopus to cancer formation may lie at the cellular, rather than at the systemic level. By comparison with the mammal, perhaps one can then see whether the two differ in particular ways, such that cancer promotors, but not their analogues, will effect their immune systems differentially.
We have studied these issues for many years, using the dramatic metamorphosis of the amphibian tadpole into the adult frog or toad. One of the principle justifications for the use of this model system to deal with these kinds of questions, is that when the adult cells develop, they turn out to be non-self in the larval body that they form in. Thus, an immune competent larva should destroy the adult cells as they are formed.
We have found that several functions of the immune system are severely compromised during metamorphosis, while other parts, e.g. the antibody- producing cells, remain normally active. This latter feature is important, because these animals would be susceptible to infectious disease and potential death during this period, if it were otherwise. Thus, the larvae make the distinction between internal (altered-self) and external (non-self) foreigness, becoming tolerant toward the first and remaining responsive to the second. Since the entire metamorphic phenomenon is stimulated and guided by changes in hormonal activity levels, our findings naturally led us into questions about the regulation of immunity by systemic or local hormones or cytokines.
We found considerable sensitivity of the immune system in metamorphic larvae (as compared to adults) to corticosteroids, e.g. cortisone, which in doses lower than physiological levels will, for instance, inhibit metamorphic plant lectin-stimulated T cell mitogenesis. We also found that corticosteroiods can inhibit the T cell immune functions which are naturally impaired during metamorphosis. Similarly, inhibition of corticosteroid synthesis in metamorphic larvae by injection of metyrapone, restores these previously impaired functions.The glucocorticoids have long been known to be effective immunomodulators and have been used for years with humans following the surgical transplantation of organs, e.g. kidney. Thus, we became concerned with how glucocorticoids affect selective immune inhibition and have studied the function of other local or systemic hormones with which they may interact. We learned that a hormone-like secretion of human activated amplifier-(helper T) immune cells, interleukin 2, which stimulates the immune response, and is inhibited by corticosteroid in human, is similarly reactive, when injected into the South African clawed toad. We used human IL-2, and human IL-1, which stimulates IL-2 production, and found that a corticosteroid will inhibit immune functions in metamorphosis by inhibiting IL-1 and therefore IL- 2 production. Thus, it seems likely that tolerance of larval cells for adult immunocytes during metamorphosis, may depend on the production of T cell anergy by the rising glucocorticoid titer. Anergy is defined as an absence of reactivity by T cells that do not produce adequate levels of IL-2. Anergy is reversible following exposure to IL-2. The larval T cells must be anergic during metamorphosis, because injection of rIL-1 or rIL-2 will restore the impaired T cell functions. Moreover, IL-1/IL-2 have the capability of stimulating immune self-destruction when injected during the metamorphic period. They may do this by breaking the immune inhibition imposed on the system by the glucocorticoid. Exogenous glucocorticoid, however, does not affect apoptosis, a form of genetically programmed cell death, in the larval thymus, as it does in the adult Xenopus or mammalian thymus. Thus, the impairment of T cell function during metamorphosis may not be through an increase in glucocorticoid-driven apoptosis during T cell development in the thymus. On the other hand, it is possible that all corticosteroid sensitized cells have already been induced to die, before the cells are set in culture to test with exogenous hormone.
The reduced repertoire of antibodies to the potential variety of foreign epitopes seen during larval development in Xenopus seems likely to be due to a limited positive selection until the late stages of metamorphosis. That is, T cells that would otherwise have been peripheralized and reactive to various epitopes, remain in the thymus to die. It is in late metamorphosis that altered-self antigen-activated apopotsis (negative clonal deletion) is first observed and interestingly, major histocompatibility complex (MHC) class I molecules are first observed. MHC Class proteins are expressed on all cells of the body and define self for each individual. In mammals, the presentation of self-immunogenic peptides depends upon MHC class I molecules. Thus, self-tolerance as a consequence of apoptosis of T cells with anti-self reactivity in early development, may depend more upon “ignorance”, that is an incapacity to recognize self antigens, and a lack of positive selection, then on negative selection. The high apoptotic rate we have seen in the thymus of early larvae may then also depend on this lack of positive selection. Cells that are not positively stimulated by antigen recognition will die by apoptosis.
Early on, we showed that one immune regulatory function, helper function or amplification, seems not to be impaired, but may actually be enhanced during metamorphosis. This suggested to us that it may be regulated by a different mechanism or that helper function is bypassed completely. We found that this function is indeed bypassed during metamorphosis. This bypass is related to the common presence of the two genetically disparate populations of immunocytes, larval and adult. They are capable of responding to each other with a mild mixed lymphocyte response (MLR) even when drawn from isogeneic strains of Xenopus. In mammalians, this type of mutual interaction of genetically disparate immune cell populations is known to lead to the production a cytokine that can activate antibody-producing cells directly and therefore, it can serve as a substitute for the presence of the kind of amplifier or helper cell cytokines, e.g. IL-2, that are normally required for immune reactivity against foreign proteins or cells. We have been able to demonstrate a cytokine of just such an activity from spleens from metamorphic, but not adult toads. Moreover, the molecule is the same size as interleukin (IL)-5, a thymus-independent (B) cell stimulator in mammals. This cytokine, sometimes called allogeneic effect factor, is able to bypass the normal adult requirement for helper function, even after thymectomy, which removes the regulatory (helper) amplifying cells. Thus, it produces a direct effect on the antibody-producing B cells.
Some years ago, in studies of their receptors on amphibian immunocytes, we established that reagents that serve to stimulate and block adrenergic receptors on mammalian cells were also effective in binding amphibian immunocytes and in modulating immune function. The adrenergic receptors are responsible for the activity of neurotransmitters and hormones, e.g. norepinephrine and epinephrine. Our data suggest that norepinephrine, which is normally present in the adult spleen, is severely reduced or absent in the spleen during metamorphosis. Since our functional studies with adults have shown that this reagent will stimulate helper T cells, but inhibit antibody-producing cells, its absence during metamorphosis would have the effect of impairing helper T cell immune activity and stimulating antibody-producing B cells. Thus, the absence of norepinephrine would have the same effect as an excess of corticosteroid. Reductions in prolactin concentration in the plasma, observed by Kikuyama in Japan, may also serve to remove a potential stimulator of immune function from the picture. PRL is now known to serve as a second messenger in IL-2 production. Similarly, the high eosinophilia observed by Per Rosenkilde of Denmark, may be a reflection of diminished parasitic/allergic reactivity and/or increased IL-5 production (eosinophil differentiating factor) in metamorphosing Xenopus.
We have established a certain degree of evolutionary conservation of IL-2 and its receptor and of IL-10. In vivo studies of IL-10 reactivity in Xenopus are planned. Earlier tests have shown that some product secreted by thymic cells of Xenopus will inhibit certain immune reactivities. Having established the evolutionary conservation of the cellular mechanisms involved in vertebrate immune regulatory events, we began to expose a potentially awesome network of interactions which functionally relate the molecular aspects of the three homeostatic mechanisms of the body, hormones, nerve secretions and the cellular products of the immune system.
However, our current focus has been on the in vitro and in vivo regulation of programmed cell death, because its regulation should have something to say about cancer susceptibility. That is, if cells die at appropriate times under normal conditions within the body, then it is unlikely that they will be in a position to become cancerous by losing their capacity to die. Programmed cell death is a normal developmental and immunological phenomenon. As noted earlier, it can be a mechanism for eliminating those immune cells with specificity to self, while T cells are developing within the thymus and B cells are developing within the spleen. Self-tolerance is the result.
We have recently found that at least two molecules used in the apoptotic process in mammalian cells are present in/on Xenopus cells. A Fas-like pro-apoptotic molecule is responsible for some induced apoptosis of Xenopus lymphocytes, although some apoptogens do not operate through the Fas pathway to caspase cascade stimulation, but instead use mitochondrial release cytochrome c to activate the caspace cascade. Receptor-induced apoptosis affects caspace 8, while mitochondrial stress reactions leading to apoptosis affect caspace 9. Phosphatidylserine (PS) is expressed on the outer surface membrane of apoptotic cells. It is recognized and bound by phagocytes which engulf the dying cells and prevent an ensuing inflammatory reaction to their contents by internalizing them. Since finding Fas and PS in Xenopus represented the first demonstrations of them in a vertebrate other than a mammal, they opened up the potential that they may reflect universal apoptotic mechanisms within the vertebrates.
Perhaps our most interesting finding recently has been, that in Xenopus cells, apoptosis can preceed DNA uptake of BUDR by hours in responses to a phorbol diester mitogen/apoptogen. Thus, it may be that, unlike the situation in mammalian cells, Xenopus cells may be able to enter apoptotic pathways without previously entering into the cell cycle. We now know, by using dual staining techniques for both apoptosis and PCNA found in dividing cells, that the cells stimulated by phorbol diesters to die are a different population than those stimulated to divide, i.e. dual staining cells are not produced in response to exposure to PMA. Thus, th Xenopus do not enter the cell cycle before dying. This could be a basis for spontaneous and induced cancer resistance in Amphibia, since cells which so easily enter apoptosis are unlikely to be transformed into cancer.
Given the potential that the regulation of apoptosis may be responsible for cancer resistance, we have turned to different aspects of apoptotic regulation to determine which differences from mammals might be responsible for the “direct” apoptosis seen in Xenopus, but not in mammals. Three features studied in 1999-2000 were 1. the potential that a permeability transition pore might be responsible for regulating mitochondrial release in glucocorticoid-induced apoptosis. Inhibition of such a mitochondrial pore inhibited apoptosis, while activation lead to apoptosis. While this was the first such demonstration in a non-mammal, the functions under study did not suggest a difference in the regulation of apoptosis, 2. The effect of IL-1/IL-2 in glucocorticoid-induction of apoptosis. Studies with mammalian thymocytes had shown that when cells are co-exposed to corticosteroid and IL-2, there was a decrease in the apoptotic level from that stimulated by the glucocorticoid alone. In Xenopus, when this was done, IL-2 did not by itself induce apoptosis, but it increased the number of cells in late stage apoptosis, suggesting that in Xenopus, that despite not initiating apoptosis by itself, IL-2 may act to drive cells more rapidly through the death process. The fact that this is an opposite result of that found with mammalian thymocytes may be a reflection of the greater requirement for mammalian cells to enter the cell cycle before dying, than has been observed with Xenopus. IL-2 is the cytokine required for T cell growth. This work needs to be repeated with larger numbers and additional study of the regulation involved. Finally, because phorbol diesters bind directly with protein kinase C, we began to study which of the PKC isoforms may be, on the one hand responsible for regulating cell division, and on the other, for regulating apoptosis, since the phenomena are at least partially separable in Xenopus, but not in mammals. We found the d isoform of PKC appeared to be particularly involved in the initiation of apoptosis, as well as, cell division. Inhibition of all isoforms with GF quickly (within three hours) blocked the capacity of PMA to stimulate both apoptosis and cell growth, while inhibition of only the Ca++-dependent isoforms with GO, failed to change control levels. Inhibition of the Ca++-independent isoforms, e.g. d, with cycloheximide or rottlerin, reduced both apoptosis and growth activated by PMA, but not as rapidly as when GF had been tested. Western assay data showed that a protein band from extracts of Xenopus cells did bind an antibody against the murine d isoform. Moreover, the band was absent when PMA-activation was affected. The band was visible, however, after PKC inhibition. Thus, it seems clear that the PKC isoform is not the crucial molecule for a stimulated cell deciding wther to die or divide, as the isoform modulated apoptosis and division in the SAME direction. Some step further down the pathway would seem to be responsible for that decision. Other questions remaining relate to the function of mitochondrial release of cytochrome c with regard to PMA-activation and inhibition, the role of Ca++ ions in this phenomenon and the proof that phosphorylation did occur when PKC was PMA-activated and was lost, when inhibited. Additionally, we have begun a study of the function of the Xenopus equivalent of p53, a factor responsible for allowing cells arrested in cycle to continue or die following disruption in their DNA by bleomycin or UV. Associated with this, is the question of what role DNA repair rates might may play in the resistance of Xenopus laevis to cancer-inducing factors. X. tropicalis is a species of Xenopus that is diploid. A comparison of repair mechanisms following bleomycin treatment showed that X. leavis was less sensitive to DNA damage and had a faster repair time than did comparable cells of X. tropicalis.Xp53 was found in both species but appeared not to be upregulated in response to UV DNA damage. Two different bands were found with X. laevis at 46 and 35 kDa but only one at 35 kDa was found with X. tropicalis.No fucntional assays have yet been made with X. tropicalis.
Reed Senior thesis students appear in Bold.
86. CLOTHIER, R.H., RUBEN, L.N., JOHNSON, R.O., PARKER, K., GREENHALGH, L., OOI, E.E., SOVAK, M. and BALLS, M. Neuroendocrine regulation of immunity: the effect of noradrenaline in Xenopus laevis. Internat'l J. Neurosci.62:123- 140 (1992).
87. RUBEN, L.N., SCHEINMAN, M., JOHNSON, R.O., SHIIGI, S., CLOTHIER, R.H. AND BALLS, M. Impaired T cell functions during amphibian metamorphosis: IL-2 receptor expression and endogenous ligand production. Mechanisms of Development 37:167-172 (1992).
88. RUBEN, L.N., RAK, J., JOHNSON, R.O., NGUYEN, N., CLOTHIER, R.H. AND SHIIGI, S.A comparison of the effects of human rIL-2 and autologous TCGF on Xenopus laevis splenocytes. Cell. Immunol.157: 300-305 (1994).
89. RUBEN, L.N., BUCHHOLZ, D., AHMADI, P.,JOHNSON, R.O., CLOTHIER, R.H. AND SHIIGI, S. Apoptosis in the thymus of adult Xenopus laevis. Dev. Comp. Immunol.18: 231-238 (1994).
90. RUBEN, L.N., AHMADI, P., BUCHHOLZ, D., JOHNSON, R.O., CLOTHIER, R.H. AND SHIIGI, S. Apoptosis in the thymus of developing Xenopus laevis. Dev. Comp. Immunol. 18:343-352 (1994).
91. RUBEN, L.N., GOODMAN, A.R., JOHNSON, R.O., KALEEBA, J., AND CLOTHIER, R.H. The development of peripheral TNP-tolerance and suppressor function in Xenopus laevis, the South African clawed toad. Dev. Comp. Immunol.19:405-415 (1995).
92. GRANT, P., CLOTHIER, R.H., JOHNSON, R.O., SCHOTT, S., AND RUBEN, L.N. The kinetics and distribution of T and B cell mitogen-stimulated apoptosis in vivo. Immunol. Letters 47:227-231 (1995).
93. RUBEN, L.N., CLOTHIER, R.H. and BALLS, M. Is the evolutionary increase in epitope-specific tolerance within the vertebrates related to cancer susceptibility ? Herpetopathol.2:99-104 (1995).
94. RUBEN, L.N., DE LEON R.T., JOHNSON, R.O. AND CLOTHIER, R.H. Interleukin-2-induced mortality during metamorphosis of Xenopus laevis. Immunol. Letts 51:157-161 (1996).
95. GRANT, P., CLOTHIER, R.H., JOHNSON, R.O. AND RUBEN, L.N. In situ lymphocyte apoptosis in larval Xenopus laevis, the South African clawed toad. Dev. Comp. Immunol.22:449-455 (1998).
96. HABERFELD, M., JOHNSON, R.O., RUBEN, L.N., CLOTHIER, R.H. AND SHIIGI, S. Adrenoceptor modulation of apoptosis in splenocytes of Xenopus laevis in vitro. (NeuroImmunoModulation 6: 175-181 (1999).
97. MANGURIAN, C., JOHNSON, R.O., McMAHAN, R., SHIIGI, S., CLOTHIER, R.H AND RUBEN, L.N. Expression of a Fas-like apoptotic molecule on larval and adult cells of Xenopus laevis. Immunol. Letts.64:31-38.
98. McMAHAN, R., JOHNSON, R.O., RUBEN, L.N. AND CLOTHEIR, R.H. Apoptosis and the cell cycle in amphibian splenic lymphocytes I. PHA and PMA exposure. Immunol. Letters 70:179-183 (1999).
99. NERA, S., VANDERBEEK, G., JOHNSON, R.O., RUBEN,L.N. AND CLOTHIER, R.H. Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African toad, as a signal for macrophage recognition Dev. Comp. Immunol. 24:641-652 (2000).
100. RUBEN, L.N., JOHNSON, R.O., BERGIN, A. AND CLOTHIER, R.H. Apoptosis and the cell cycle in Xenopus laevis: PMA and OMPMA exposure of thymocytes and splenocytes Apoptosis 5:225-234 (2000).
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