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Age-Induced Hair Graying and Oxidative Stress

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Abstract

Age-induced hair graying (canities), or the age-induced loss of melanin synthesis and deposition within the hair shafts, is a noticeable and undesired sign of the aging process. Numerous mechanisms contribute to age-induced hair graying, affecting both follicular and stem cell melanocytes and acting at different follicular locations. Many of these processes are induced, directly or indirectly, by oxidative insults and damage. Melanin-producing bulbar melanocytes express high levels of BCL-2 to survive reactive oxygen species (ROS) attacks, which are induced by the melanogenic process itself and by ultraviolet A (UVA) irradiation. With aging, the expression of BCL-2, and possibly of TRP-2, is reduced, and the endogenous, enzymatic antioxidant defense system declines, resulting in greater oxidative stress. In particular, catalase expression and activity are markedly reduced with aging, leading to millimolar accumulation of hydrogen peroxide within the hair follicle and contributing to bulbar melanocyte failure and death. Additionally, exposure of melanocyte stem cells to cumulative oxidative damage, combined with reduced BCL-2 protective levels, results in apoptosis and therefore decreases the number of melanocytes that could repopulate the newly formed anagen follicles. Altogether, oxidative stress may contribute to age-induced hair graying via multiple pathways. Better understanding of the different processes, sources, and types of oxidative stress within the follicular environment, and the different susceptibilities of melanocytes to oxidative stress at the different follicular locations, might yield clues to possible interventions for prevention or reversal of hair graying.

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Skin and hair phenotypes are powerful cues in human communication. They impart much information, not least about our racial, ethnic, health, gender and age status. In the case of the latter parameter, we experience significant change in pigmentation in our journey from birth to puberty and through to young adulthood, middle age and beyond. The hair follicle pigmentary unit is perhaps one of our most visible, accessible and potent aging sensors, with marked dilution of pigment intensity occurring long before even subtle changes are seen in the epidermis. This dichotomy is of interest as both skin compartments contain melanocyte subpopulations of similar embryologic (i.e., neural crest) origin. Research groups are actively pursuing the study of the differential aging of melanocytes in the hair bulb versus the epidermis and in particular are examining whether this is in part linked to the stringent coupling of follicular melanocytes to the hair growth cycle. Whether some follicular melanocyte subpopulations are affected, like epidermal melanocytes, by UV irradiation is not yet clear. A particular target of research into hair graying or canities is the nature of the melanocyte stem compartment and whether this is depleted due to reactive oxygen species-associated damage, coupled with an impaired antioxidant status, and a failure of melanocyte stem cell renewal. Over the last few years, we and others have developed advanced in vitro models and assay systems for isolated hair follicle melanocytes and for intact anagen hair follicle organ culture which may provide research tools to elucidate the regulatory mechanisms of hair follicle pigmentation. Long term, it may be feasible to develop strategies to modulate some of these aging-associated changes in the hair follicle that impinge particularly on the melanocyte populations.
Article
Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway. The oncogenic potential of Bcl-2 is well established, with its overexpression reported in various cancers. The antiapoptotic function of Bcl-2 is closely associated with its expression levels. Reactive oxygen and nitrogen species (ROS/RNS) are important intracellular signaling molecules that play a key role in various physiological processes including apoptosis. We have recently reported that ROS and RNS can regulate Bcl-2 expression levels, thereby impacting its function. Superoxide anion (*O(2)(-)) plays a proapoptotic role by causing downregulation and degradation of Bcl-2 protein through the ubiquitin-proteasomal pathway. In contrast, nitric oxide (NO)-mediated S-nitrosylation of Bcl-2 prevents its ubiquitination and subsequent proteasomal degradation, leading to inhibition of apoptosis. Interestingly, NO-mediated S-nitrosylation and stabilization of Bcl-2 protein was the primary mechanism involved in the malignant transformation of nontumorigenic lung epithelial cells in response to long-term carcinogen exposure. We describe a novel mechanism of Bcl-2 regulation by *O(2)(-) and NO, providing a new dimension to reactive species-mediated Bcl-2 stability, apoptotic cell death, and cancer development.
Article
Dopachrome tautomerase (Dct) is a critical enzyme in the melanogenesis pathway that isomerizes the intermediate dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and influences the proportion of DHICA monomer incorporated into the 5,6-dihydroxyindole (DHI) polymer in eumelanin. To investigate whether Dct inactivation affects skin photoprotection against ultraviolet radiation, we examined levels of reactive oxygen species (ROS), sunburn cell formation, epidermal cell apoptosis, and melanin composition in skins of Dct(-/-) knockout mice compared with skins of wild-type C57BL/6 mice under UVA-induced oxidative stress. The results demonstrate that Dct inactivation elevates the level of ROS, increases the numbers of sunburn cells and apoptotic cells, and decreases the amount of eumelanin in the epidermis upon exposure to chronic UVA radiation. Moreover, we determined the effects of DHICA-melanin, DHI-melanin, and a mixture of both on hydroxyl radical generation in the Fenton reaction utilizing an electron spin resonance assay. DHICA-melanin exhibits a potent hydroxyl radical-scavenging activity, whereas DHI-melanin does not. Thus, this study suggests that DHICA monomers are required to incorporate into the DHI polymer backbone of eumelanin, which highlights the important role of Dct in the regulation of DHICA-mediated antioxidation.
Article
Few biological data on human eyelash follicles have been reported in the literature. To characterize eyelash follicle growth, cycle and morphology, and further investigate the biological mechanisms that determine eyelash length, curl and pigmentation, compared with scalp hair follicle. Twenty-nine caucasian female volunteers aged between 26 and 60 years were enrolled in the study to provide eyelashes. Four of these volunteers were followed weekly for 9 months to characterize their eyelash cycle. Eyelash length and time of renewal were measured using a high-resolution camera and image analysis. Immunohistological study of the bulbs were performed on eyelid biopsies from 17 patients requiring block excision for ectropion repair. The calculated durations of anagen phase and complete cycle of the eyelashes were 34 + or - 9 and 90 + or - 5 days, respectively. Eyelash follicle growth rate was quite variable, with an average rate of 0.12 + or - 0.05 mm daily. Eyelash follicle morphology was very close to that of the scalp hair follicle, but some remarkable differences were noticed. For example, the K19-positive epithelial stem cell population was spread all along the follicle and not split into two reservoirs as seen in scalp hair follicles. Some asymmetry was detected in HSPG and CSPG, as well as K38 (formerly Ha8) and K82 (formerly Hb2) distribution, similar to that observed in curly hair. Finally, dopachrome tautomerase was found expressed in eyelash follicle melanocytes, while it was strikingly absent in scalp hair follicle melanocytes. The eyelash is structurally very close to curly hair but some biological processes related to follicle cycle and pigmentation differ markedly.
Article
The 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside (THSG), a water-soluble active component extracted from dried tuber root of Polygonum multiflorum, has been found to induce pigmentation in B16 cells, but the details of the underlying mechanism remain unknown. The present study was conducted to investigate the mechanism of stimulatory effect of THSG on melanogenesis using B16F1 melanoma cells. Several experiments were performed in B16F1 melanoma cells. We studied melanin content, tyrosinase activity, cell viability, and performed reverse transcription polymerase chain reaction and Western blots for proteins involved in melanogenesis. THSG increased the melanin content and tyrosinase activity in a concentration-dependent manner and treatment with 10 microg/ml THSG enhanced the expression of tyrosinase time-dependently in B16 cells. We then investigated whether THSG influences the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. THSG was found to induce sustained MITF up-regulation and cAMP response element (CRE) binding protein (CREB) activation, suggesting that THSG-mediated MITF activation may be cAMP dependent. Furthermore, Western blot analysis revealed that THSG elevated the level of phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) significantly at 1-6 h; a p38 MAPK inhibitor, SB203580, almost completely attenuated the THSG-mediated up-regulation of melanin synthesis and induction of MITF and tyrosinase expression. THSG exerts its stimulatory effect on melanogenesis by MAP kinase activation and MITF-induction of tyrosinase.
Article
Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT-Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H(2)O(2)) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met-S=O) repair in the entire gray hair follicle. Accordingly, Met-S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT-Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L-methionine. In summary, our data feed the long-voiced, but insufficiently proven, concept of H(2)O(2)-induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.
Article
Skin and hair colour contribute significantly to our overall visual appearance and to social/sexual communication. Despite their shared origins in the embryologic neural crest, the hair follicle and epidermal pigmentary units occupy distinct, although open, cutaneous compartments. They can be distinguished principally on the basis of the former's stringent coupling to the hair growth cycle compared with the latter's continuous melanogenesis. The biosynthesis of melanin and its subsequent transfer from melanocyte to hair bulb keratinocytes depend on the availability of melanin precursors and on a raft of signal transduction pathways that are both highly complex and commonly redundant. These signalling pathways can be both dependent and independent of receptors, act through auto-, para- or intracrine mechanisms and can be modified by hormonal signals. Despite many shared features, follicular melanocytes appear to be more sensitive than epidermal melanocytes to ageing influences. This can be seen most dramatically in hair greying/canities and this is likely to reflect significant differences in the epidermal and follicular microenvironments. The hair follicle pigmentary unit may also serve as an important environmental sensor, whereby hair pigment contributes to the rapid excretion of heavy metals, chemicals and toxins from the body by their selective binding to melanin; rendering the hair fibre a useful barometer of exposures. The recent availability of advanced cell culture methodologies for isolated hair follicle melanocytes and for intact anagen hair follicle organ culture should provide the research tools necessary to elucidate the regulatory mechanisms of hair follicle pigmentation. In the longer term, it may be feasible to develop hair colour modifiers of a biological nature to accompany those based on chemicals.
Article
WE have previously suggested that factors which regulate the proliferation and pigmentation of Cloudman S-91 mouse melanoma cells share common biochemical pathways1,2. Our conclusion was based on the following observations: (1) most mutant cells selected for their ability to grow in culture medium containing melanocyte-stimulating hormone (MSH) show an altered ability to produce pigment1, and (2) tyrosinase is activated in response to elevated levels of cyclic AMP in a reaction apparently mediated by a cyclic AMP-dependent protein kinase3. This same protein kinase has been implicated as a positive regulator in the proliferation of Cloudman cells4. In addition, we found that some precursors of melanin are cytotoxic and that cells actively producing melanin run the risk of self-destruction through a build-up of these precursors. We have previously shown that pigmented cells are killed as a result of exposure to excess tyrosine or dihydroxyphenylalanine (dopa) in the culture medium2,5. It has since been demonstrated that actively melanising cells release a cytotoxic agent into their culture medium6. Here we present further evidence for the cytotoxicity of melanin precursors and show that 5,6-dihydroxyindole is one of the toxic compounds in the melanin biosynthetic pathway.
Article
In order to determine whether amelanotic melanocytes are present in senile white hair, we attempted to detect tyrosinase mRNA and its protein using in situ hybridization and immunohistochemical staining. Specifically stained cells containing tyrosinase mRNA or its protein were detected by each technique in 7/23 (30.4%) or 4/14 (28.6%) senile white hairs, respectively. These cells were located in the outer root sheath between the hair-bulb and the infundibulum. Silver staining was used to determine whether melanin was present within the outer root sheath of the senile white hair. Because none of the 21 white hairs tested showed specific staining within the outer root sheath, the absence of melanin was confirmed. Thus our results suggest the presence of amelanotic melanocytes within the outer root sheath of senile white hair.
Article
The maintenance of homoeostasis in normal tissues reflects a balance between cell proliferation and cell death. The importance of both positive and negative regulators of cell growth has been well documented in neoplasia. bcl-2 argues for the existence of a new category of oncogenes, regulators of programmed cell death. The bcl-2 gene was identified at the chromosomal breakpoint of t(14;18) bearing B cell lymphomas. Bcl-2 has proved to be unique among proto-oncogenes in being localized to mitochondria and in blocking programmed cell death rather than affecting proliferation. In adults, bcl-2 is topographically restricted to progenitor cells and long-lived cells in tissues characterized by apoptotic cell death. Bcl-2 is confined to the zones of surviving B cells in germinal centres. Within thymus, bcl-2 is present in the surviving mature thymocytes of the medulla but absent from the majority of immature cortical thymocytes, most of which die by apoptosis. Transgenic mice that overexpress bcl-2 in the B cell lineage demonstrate extended cell survival and prolonged immune responses and indicate a role for bcl-2 in B cell memory. Transgenic models that overexpress bcl-2 in the thymus have expanded the involvement of bcl-2 to multiple apoptotic pathways and indicate its involvement in thymocyte maturation. Moreover, the development of tumours in transgenic mice that overexpress bcl-2 indicates the potential importance of oncogenes in interfering with programmed cell death. Alterations in genes that regulate cell death may prove to be key events in neoplasia, extending the life span of cells and thus increasing their opportunity to acquire additional genetic aberrations.
Article
In C57 Bl-6 mice, melanogenesis is strictly coupled to the growth phase of the hair cycle (anagen). To further study this phenomenon of concerted developmental and pigmentary activity, we followed the sequence of tyrosinase (key enzyme of melanogenesis) expression and activity and the presence of the melanosomal protein gp 75 during the development of traumatically induced anagen follicles (days 0 = telogen, and days 1-12, after anagen induction studied). In addition to performing Northern and Western blots for tyrosinase, tyrosine hydroxylase activity (THA) and dopa oxidase activity (DOA) were measured. On day 0, DOA was undetectable, and THA was very low. On days 1 and 2, both activities were undetectable; starting from day 3, they increased rapidly, reaching a plateau on days 8 and 12. DO-positive proteins had apparent molecular weights (MW) of 66-68 kD (days 3-12), 72-74 kD (days 5-12), and 130 kD (days 8 and 12). Western blotting emphasized proteins of MW 66-68 kD (tyrosinase), and 73-75 kD (gp 75); tyrosinase was undetectable on day 0, but already present on days 1 and 2; it increased by day 5 and had reached a plateau on days 8 and 12; gp 75 was undetectable on days 0-2; it was present on day 3, increased by day 5, and reached a plateau on days 8 and 12. Northern blot analysis revealed high levels of tyrosinase mRNA on days 5 and 8, low levels on days 1-3, and none on day 0. These data suggest a highly regulated, time frame-restricted, differential pattern of tyrosinase transcription, translation, and enzyme activity during the different stages of the developing murine anagen follicle, possibly as a result of complex interactions between follicular melanocytes and their environment.
Article
The hydrogen peroxide produced during the autoxidation of melanin pigments has been measured using an oxidase electrode. The autoxidation has been shown to occur via the superoxide intermediate. The melanin pigment competes with superoxide dismutase for the scavenging of superoxide radicals. However, superoxide dismutase at high concentrations caused a substantial increase in the production of hydrogen peroxide, formed during melanin autoxidation. The implications of this finding are discussed in light of melanin's ability to function as a pseudo-dismutase.
Article
Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin.
Article
GREYING of hair is probably the most obvious sign of ageing in man. To determine whether or not its onset and progress are influenced by hair colour we have examined a sample of Australians.
Article
The maintenance of homeostasis in normal tissues reflects a balance between cell proliferation and cell death. Bcl-2 inaugurated a new category of oncogenes, regulators of cell death. The Bcl-2 gene was identified at the chromosomal breakpoint of t(14;18) bearing B cell lymphomas. Bcl-2 proved unique by blocking programmed cell death rather than promoting proliferation. In adults, Bcl-2 is topographically restricted to progenitor cells and longlived cells but is much more widespread in the developing embryo. Transgenic mice that overexpress Bcl-2 demonstrate extended cell survival, and progress to high grade lymphomas. Bcl-2 has been localized to mitochondria, endoplasmic reticulum and nuclear membranes, also the sites of reactive oxygen species generation. Bcl-2 does not appear to influence the generation of oxygen free radicals but does prevent oxidative damage to cellular constituents including lipid membranes. Bcl-2 deficient mice complete embryonic development but undergo fulminant lymphoid apoptosis of thymus and spleen. Moreover, they demonstrate two unexpected pathologies resulting from cell death, polycystic kidney disease and hair hypopigmentation. The latter is a potential oxidant injury from the melanin biosynthetic pathway. A family of Bcl-2 related genes is emerging that includes Bax, a conserved homolog that heterodimerizes in vivo with Bcl-2 and promotes cell death. The ratio of family members, such as Bcl-2/Bax, determines the survival or death of cells following an apoptotic stimulus.
Article
Mammalian melanocytes can produce two basic types of melanin, eumelanin and pheomelanin, within discrete organelles termed melanosomes. The physiological signals that regulate this switch are extrinsic to the melanocyte, and include alpha-melanocyte stimulating hormone and the agouti protein. Tyrosinase, encoded at the albino locus, is the enzyme essential for the synthesis of both types of melanin, but other tyrosinase-related proteins (e.g. TRP1 encoded at the brown locus and TRP2 encoded at the slaty locus) regulate eumelanogenesis catalytically at steps distal to tyrosinase (as 5,6-dihydroxyindole-2-carboxylic acid oxidase and DOPAchrome tautomerase, respectively). The silver protein is another melanosomal protein, and although it has some limited homology to the tyrosinase-related proteins, it does not have any known enzymatic function and probably serves as a structural matrix protein. The role of each of those melanosomal proteins in pheomelanogenesis, however, is still unclear. In this study, we have compared the expression and catalytic functions of those proteins in pheomelanic and eumelanic hair bulb melanocytes. There was no detectable expression of TRP1 or TRP2, or either of their enzymatic activities, in hair bulbs of lethal yellow (Ay/a) newborn mice, and tyrosinase activity was present at a reduced level compared to that found in hair bulbs of black (a/a) newborn mice. Similar results were observed in regenerating hair bulbs of adult lethal yellow mice and in hair bulbs of 5- to 7-day-old agouti mice (A/A), an age where pheomelanin is produced predominantly. Expression of the silver protein was similarly not observed in hair bulbs of the pheomelanic mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
The bcl-2 gene was initially identified through its participation in the translocation 14:18 in B-cell lymphomas of follicular center cell origin. This classic translocation juxtaposes the transcriptionally active immunoglobulin heavy-chain locus on chromosome 14 to the bcl-2 gene on chromosome 18, resulting in overexpression of bcl-2 protein. The oncogene bcl-2 is thought to prolong cell survival through interference with programmed cell death. To date, bcl-2 expression has been reported in normal lymphoid, hemopoietic, neural, breast and prostatic tissues. Since melanocytes are neural crest derivatives and have an extended life-span, our objective was to determine whether the bcl-2 protein is expressed in human melanocytes. We analyzed normal skin biopsies for bcl-2 expression using standard immunohistochemistry. As hypothesized, dendritic cells in the basal epidermal layer stained strongly for bcl-2. The distribution and morphology of these cells was typical for melanocytes. Additionally, eccrine sweat glands, lymphocytes and the dermal papilla of hair follicles demonstrated bcl-2 positivity. We believe this to be the first report of bcl-2 expression in normal melanocytes.
Article
The hair follicle undergoes a cycle of growing, regressing, and resting phases (anagen, catagen, telogen, respectively). As the follicle enters catagen, the cells of the lower, cycling portion undergo a process of controlled cell death (apoptosis). Understanding the mechanism of apoptosis in the follicle should give insight into one of the control steps of hair cycling. In this study we sought the expression of bcl-2, a protooncogene associated with apoptosis control, in the cycling follicle of the adult mouse. Using a monoclonal antibody to the mouse protein we immunolocalized bcl-2 gene product in the cycling pelage follicle of the C57/B6 adult mouse. The protein was expressed in the follicular papilla (a non-cycling portion of the follicle) throughout the cycle-including telogen. The cycling follicular epithelium, however, showed positive antibody staining in anagen, which decreased in catagen and disappeared in telogen. In anagen the cells of the bulb, bulge, and basal layer of the outer root sheath expressed the bcl-2 protein. Understanding the action of this apoptosis-inhibiting molecule should serve to elucidate the dynamics of follicular cycling.
Article
Slaty (slt), an autosomal recessive mutation that arose in YZ57/Ch mice, results in the dilution of coat color and premature hair loss. Recently, a gene encoding a homologue of the melanogenic enzyme tyrosinase (termed tyrosinase related protein 2 or TRP2) was cloned and was subsequently mapped to the slaty locus on chromosome 14. TRP2 was shown to function in melanogenesis as DOPAchrome tautomerase by means of the catalytic activity of the immunopurified protein and in slaty mutant skin samples. In this study, we generated an expression vector of a murine TRP2 encoding cDNA and are able to confirm its activity as DOPAchrome tautomerase. We further demonstrate that the slaty mutation dramatically decreases the catalytic function of the protein.
Article
The maintenance of homeostasis in normal tissues reflects a balance between cell proliferation and cell death. The importance of both positive and negative regulators of cell growth has been well documented in neoplasia. Bcl-2 argues for the existence of a new category of oncogenes, regulators of cell death. The bcl-2 gene was identified at the chromosomal breakpoint of t(14; 18) bearing B cell lymphomas. Bcl-2 has proved to be unique among protooncogenes in blocking programmed cell death rather than promoting proliferation. In adults, bcl-2 is topographically restricted to progenitor cells and longlived cells but is much more widespread in the developing embryo. Transgenic mice that overexpress bcl-2 in the B cell lineage demonstrate extended cell survival, and progress to high grade lymphomas. Bcl-2 has been localized to mitochondria, endoplasmic reticulum and nuclear membranes, also the sites of reactive oxygen species generation. Bcl-2 does not appear to influence the generation of oxygen free radicals but does prevent oxidative damage to cellular constituents including lipid membranes. Bcl-2 deficient mice complete embryonic development and display relatively normal haematopoietic differentiation but undergo fulminant lymphoid apoptosis of thymus and spleen. Moreover, they demonstrate two potentially oxidation related pathologies: polycystic kidney disease and hair hypopigmentation. A family of bcl-2 related genes is emerging that includes Bax, a conserved homolog that heterodimerizes in vivo with bcl-2. A pre-set ratio of Bcl-2/Bax appears to determine the survival or death of cells following an apoptotic stimulus.
Article
Mice carrying ablated coding regions of the bcl-2 alpha and bcl-2 beta transcripts have been made. bcl-2-/- mutants are smaller but viable, although about half of them die by 6 weeks of age. As shown earlier with somatic bcl-2 gene-targeted mice, the number of lymphocytes markedly decreased within few weeks after birth while other hematopoietic lineages remained unaffected. Among lymphocytes, CD8+ T cells disappeared most quickly followed by CD4+ T cells, whereas B cells were least affected. bcl-2-/- lymphocytes, however, could respond normally to various stimuli including anti-CD3, Con A, phorbol 12-myristate 13-acetate plus ionomycin, interleukin 2, lipopolysaccharide, and anti-IgM antibody. Abnormalities among nonlymphoid organs include smaller auricles, hair color turning gray at 4-5 weeks of age, and polycystic kidney disease-like change of renal tubules. These results suggest that Bcl-2 may be involved during morphogenesis where inductive interactions between epithelium and mesenchyme are important such as in the kidneys, hair follicles, and perichondrium of auricles. Surprisingly, the nervous system, intestines, and skin appear normal despite the fact that these organs show high levels of endogenous Bcl-2 expression in normal mice.