Sensitivity to sunburn is associated with susceptibility to ultraviolet radiation-induced suppression of cutaneous cell-mediated immunity.

Skin cancer incidence is highest in white-skinned people. Within this group, skin types I/II (sun sensitive/tan poorly) are at greater risk than skin types III/IV (sun tolerant/tan well). Studies in mice demonstrate that ultraviolet radiation (UVR)-induced suppression of cell-mediated immune function plays an important role in the development of skin cancer and induces a susceptibility to infectious disease. A similar role is suspected in humans, but we lack quantitative human data to make risk assessments of ambient solar exposure on human health. This study demonstrates that ambient levels of solar UVR, typically experienced within 1 h of exposure to noonday summer sunlight, can suppress contact hypersensitivity (CHS) responses in healthy white-skinned humans in vivo (n = 93). There was a linear relationship between increase in erythema and suppression of CHS (P < 0.001), and a moderate sunburn (two minimal erythema doses [2 MED]) was sufficient to suppress CHS in all volunteers by 93%. However, a single suberythemal exposure of either 0.25 or 0.5 MED suppressed CHS responses by 50 and 80%, respectively, in skin types I/II, whereas 1 MED only suppressed CHS by 40% in skin types III/IV. The two- to threefold greater sensitivity of skin types I/II for a given level of sunburn may play a role in their greater sensitivity to skin cancer.

Tumor necrosis factor alpha is a critical component of interleukin 13-mediated protective T helper cell type 2 responses during helminth infection.

In vivo manipulation of cytokine and/or cytokine receptor expression has previously shown that resistance to infection with the caecum-dwelling helminth Trichuris muris is dependent on interleukin (IL)-4 and IL-13 while susceptibility is associated with a T helper cell type 1 (Th1) cytokine response. Using gene-targeted mice deficient in tumor necrosis factor (TNF) receptor signaling and anti-TNF-alpha monoclonal antibody treatment, we have extended these studies to reveal a critical role for TNF-alpha in regulation of Th2 cytokine-mediated host protection. In vivo blockade of TNF-alpha in normally resistant mice, although not altering IL-4, IL-5, or IL-13 production in the draining lymph node, significantly delayed worm expulsion for the duration of treatment. IL-13-mediated worm expulsion in IL-4 knockout (KO) mice was also shown to be TNF-alpha dependent, and could be enhanced by administration of recombinant TNF-alpha. Furthermore, TNF receptor KO mice failed to expel T. muris, producing high levels of parasite-specific immunoglobulin G2a and the generation of a predominantly Th1 response, suggesting that the absence of TNF function from the onset of infection dramatically alters the phenotype of the response. These results provide the first demonstration of the role of TNF-alpha in regulating Th2 cytokine-mediated responses at mucosal sites, and have implications for the design of rational therapies against helminth infection and allergy.

Early and late incorporation of tritiated thymidine into skin cells and the presence of a long-lived G 0 -specific precursor pool.

40 min after a single injection of 50 microCi of tritiated thymidine a 3 mm punch of DBA-1 mouse skin contains about 1000 dpm. This value remains constant for at least 48 hr after injection. 50 hair follicles contain about 40 dpm, and from these values the activity calculated to reside in the basal layer of a 3 mm punch of skin is 760 dpm. These values also remain constant with time after injection. Fresh punches of skin contain much more activity. The fixative-soluble fraction (the difference between fresh and fixed values) decays slowly with time. The values for DBA-2 mice are similar. Plucking the hair from the follicles appears immediately to increase the size of the fixative-soluble fraction and decrease the fixed tissue values to about 500 dpm per punch for whole skin and about 1 dpm per 50 follicles for DBA-1. Thus almost all the activity is restricted to the epidermis. The fixative-soluble fraction returns approximately to the unplucked value between 24 and 48 hr after plucking. However, during this period the fixed tissue values are rising rapidly as stimulated cells enter S. It appears that in both strains labeled material remains available for incorporation into stimulated cells for at least 48 hr after a single injection. The amount persisting appears to decrease with time. The whole-fixed skin, the hair follicles, and the epidermis all contain cells that are capable of becoming labeled after stimulation 8-48 hr after an injection. The label in question does not become incorporated into normal cycling skin or hair follicle cells. It is concluded that the DNA precursor pool is possibly connected with G(0) cells and that both the hair follicle and the basal layer of the epidermis contain these resting cells.

Labeling of murine mastocytoma cells in vitro with plasma tritiated thymidine-labeled animals.

40 min after injecting tritiated thymidine into an animal, 20-30% of the total plasma radioactivity is nonvolatile. This fraction decreases to about 6% 10 hr after the injection and 3% 24 hr after the injection. There appears to be material in this nonvolatile fraction that can label mastocytoma cells in culture. The labeling indices decrease with time after injection in the same way as the nonvolatile fraction. The 40 min plasma sample contains sufficient material to allow accurate assessment of the fraction of cells in S in culture after a 6 wk exposure. The circulating material is not apparently available for incorporation into those cells in cycle in the donor animal. The material appears to be related to the G(0) cell-specific pool that has been described elsewhere. The trichloroacetic acid-soluble or ethanol-soluble nonvolatile activity appears to contain thymine, and some thymidine-phosphorylated compounds.

Assessment of hormone dependence of comedo ductal carcinoma in situ of the breast.

BACKGROUND: Ductal carcinoma in situ (DCIS) represents 20%-30% of breast cancers detected by clinical screening (i.e., mammography). More than 50% of DCIS lesions may be estrogen receptor negative and, therefore, hormone independent. However, the role of estrogen in the natural history of DCIS is unknown. PURPOSE: A novel in vivo (i.e., xenograft) model was developed to determine to what degree DCIS lesions depend on estrogen for growth. METHODS: Specimens of breast tissue were collected from 52 women during diagnostic or therapeutic surgical procedures. Portions of each specimen were randomly selected and analyzed by histology and thymidine labeling (to measure cell proliferation). The remainder of each specimen was implanted into five to 18 athymic BALB/c nu/nu mice (depending on the amount of tissue available), with eight pieces of approximately 2 mm x 2 mm x 1 mm implanted at different locations on the back of each mouse. Half of the mice received implants containing estrogen (2 mg 17 beta-estradiol), and the other half received placebo implants. Levels of cell proliferation in xenografts, recovered after 14, 28, 42, or 56 days in the mice, were measured by thymidine labeling or by immunohistochemistry through use of an antibody specific for the Ki-67 nuclear antigen. Immunohistochemistry was also used to measure the levels of estrogen receptor in the tissue specimens. Serum 17 beta-estradiol levels in the mice were measured by radioimmunoassay. RESULTS: Initial levels of cell proliferation were approximately 10-fold higher in 10 specimens with estrogen receptor-negative, comedo (i.e., more malignant in appearance) DCIS than in four specimens with estrogen receptor-positive DCIS (mean proliferation indices: 22% versus 1.9%, respectively; two-sided P < .001). Xenografts from the majority of specimens survived up to 56 days in the mice and maintained good architectural and cellular preservation. Estrogen treatment of the xenograft-bearing mice had no effect on the high level of cell proliferation observed in estrogen receptor-negative, comedo DCIS specimens (two-sided P = .89). In contrast, increased levels of cell proliferation in response to estrogen supplementation were measured in three estrogen receptor-positive, noncomedo DCIS specimens (two-sided P < .001). However, even with estrogen treatment, cell proliferation levels in estrogen receptor-positive DCIS specimens did not reach those seen in estrogen receptor-negative DCIS specimens. CONCLUSION AND IMPLICATION: Estrogen receptor-negative, comedo DCIS lesions appear to be estrogen independent; therefore, antiestrogen (e.g., tamoxifen) therapy may not benefit patients with comedo DCIS.

1,2-Dimethylhydrazine-induced colon carcinoma and lymphoma in msh2(-/-) mice.

BACKGROUND: Defective mismatch repair (MMR) in humans is particularly associated with familial colorectal cancer, but defective repair in mice is generally associated with lymphoma in the absence of experimental exposure to carcinogens. Loss of MMR also confers resistance to the toxic effects of methylating agents. We investigated whether resistance to methylation contributes to increased susceptibility to colorectal cancer in mice by exposing mice with defects in the MMR gene msh2 to a methylating agent. METHODS: Tumor incidence and time of death in msh2(+/+), msh2(+/-), and msh2(-/-) mice were analyzed after weekly exposure (until tumor appearance) to the methylating agent 1,2-dimethylhydrazine (DMH). Chemically induced and spontaneous tumors were characterized by frequency, type, and location. The tumor incidence in untreated and treated mice of each genotype was compared by a Mann-Whitney U test. Carcinogen-induced apoptosis in histologic sections of small and large intestines was also determined. All statistical tests were two-sided. RESULTS: Homozygous inactivation of the msh2 gene statistically significantly accelerated (P<.0001) death due to the development of DMH-induced colorectal tumors and lymphomas. Rates of death from DMH-induced colorectal adenocarcinoma were similar in msh2 heterozygous and wild-type mice, but only msh2 heterozygotes (msh(+/-)) developed additional, noncolorectal malignancies (notably trichofolliculoma [two of 21], angiosarcoma of the kidney capsule [two of 21], and lymphoma [one of 21]), suggesting that heterozygosity for msh2 slightly increases DMH susceptibility. DMH induced apoptosis in small intestinal and colonic epithelial crypts that was dependent on active msh2. CONCLUSIONS: Inactivation of msh2 allows the proliferation of gastrointestinal tract cells damaged by methylating agents. Furthermore, MMR constitutes a powerful defense against colorectal cancer induced by DNA methylation.

Adult small intestinal stem cells: identification, location, characteristics, and clinical applications.

There are few systems which enable adult tissue stem cells to be studied. However, the gastrointestinal tract with its high degree of polarity, well-defined cell migratory pathways, and dynamic cell replacement is a model tissue providing unique opportunities for stem cell study. Lineage tracking indicates that all cell replacement originates at well-defined stem cell positions, with an associated slower cell cycle. Radiobiological studies suggest a hierarchical stem cell compartment (actual and potential stem cells). Actual stem cells have an intolerance of genotoxic damage and die via apoptosis. Stem cells also selectively sort the old and new DNA strands at division, retaining the replication error free strands in the stem cell daughter. High genotoxic sensitivity and selective sorting of old and new DNA strands, provides extremely effective protective mechanisms against both replication and random errors. This provides a new explanation for the low cancer risk in the small intestine.

The effect of exogenous prostaglandin administration on tumor size and yield in Min/+ mice.

This study set out to examine the effect of exogenous prostaglandin (PG) administration on tumor development in Min/+ mice. Mice were treated with the stable prostaglandin E2 analogue 16,16-dimethyl-PGE2 from 6-18 weeks of age. Mice were sacrificed, and tumor burden was assessed using morphometric techniques. Parameters measured were median tumor size, mean tumor size, the proportion of the area of the gastrointestinal mucosa covered with tumor, and the number of tumors per 1000 mm2 of gastrointestinal mucosa. In addition, proliferative and apoptotic indices were determined. These measurements were carried out for all regions of the small intestine (i.e., duodenum, jejunum, upper ileum, and lower ileum) and the large intestine (i.e., cecum and mid-colon/rectum). 16,16-Dimethyl-PGE2-treated animals showed a significant decrease in tumor burden (by approximately 50-70%), in comparison with those animals that were treated with vehicle alone (0.001% ethanol in 0.9% sterile saline), in all regions of the intestine (at P = 0.008 or better). This effect was contributed to by a reduction in the number of tumors (by approximately 20-50%) and a reduction in tumor size (by approximately 10-70%). An increase in tumor cell turnover was associated with this decrease in tumor burden, as determined by the changes in the levels of thymidine incorporation (significant at P = 0.003), apoptosis, and mitosis (nonsignificant).

Inhibition of doxorubicin-induced apoptosis in vivo by 2-deoxy-D-glucose.

Previous studies have shown that DNA cleavage by mammalian topoisomerase II is ATP dependent and can be inhibited by metabolic inhibitors. Furthermore, it has been shown that metabolic inhibitors also have a cytoprotective effect in vitro against topoisomerase II-targeting antitumor drugs. However, the nature of the ATP-dependent process is not known. We have previously shown that doxorubicin induces apoptosis (programmed cell death) in the murine small intestine which can be inhibited by the protein synthesis inhibitor cycloheximide. In the present study, we have demonstrated that 2-deoxy-D-glucose reduces the incidence of doxorubicin-induced apoptosis in vivo if administered within 45 min of the doxorubicin. Maximum reduction was observed at 2 h after treatment (approximately 66%); however, significant reduction was still observable at 9 h after treatment (approximately 33%). Significant positive correlation was observed between protein synthesis inhibition and apoptosis inhibition. Other possible mechanisms of action of the inhibitor do not appear to be important in cytoprotection. The inhibitor did not reduce the uptake of doxorubicin into the intestinal epithelium; however, it caused a significant increase in retention of the drug. The kinetics of inhibition suggest that alteration of cell cycle kinetics, inhibition of formation of doxorubicin-topoisomerase II complex or induction of glucose-regulated proteins are not significant factors in cytoprotection. These studies indicate that at least in the mouse small intestinal epithelium, the ATP-dependent process in cell killing by doxorubicin may involve protein synthesis.

The relationships between p53-dependent apoptosis, inhibition of proliferation, and 5-fluorouracil-induced histopathology in murine intestinal epithelia.

The relationship between acute (<36 h) induction of apoptosis and longer-term (>72 h) intestinal histopathology was systematically investigated in vivo using p53 wild-type (+/+) and null (-/-) mice. Administration of the enterotoxin 5-fluorouracil (5-FU) at either 40 or 400 mg/kg to BDF1 mice induced an acute p53-dependent apoptosis in the crypts of both small intestine and midcolon. Although the amount of apoptosis was of the same order of magnitude at its peak (24 h) at both doses, only 400 mg/kg 5-FU brought about histopathological changes to the gut after 96 h, quantified as losses of crypt and villus cellularity. Only after the administration of 400 mg/kg 5-FU were mitotic index and DNA synthesis significantly suppressed in both small intestinal and midcolonic crypts at 24 h. This correlated with a prolonged, p53-dependent expression of p21waf-1/cip1. In p53 null (-/-) mice, significant reductions in both 5-FU-induced apoptosis and inhibition of cell cycle progression allowed retention of crypt integrity 96 h after 5-FU. These results show that quantitative measures of acute apoptosis in vivo may not accurately predict subsequent pathological changes in the gut. Rather, p53-dependent inhibition of cell cycle progression, together with cell loss by apoptosis, caused a loss of crypt integrity. Importantly, the tissue toxicity of 5-FU was genetically determined at a locus (p53) separate from that directly associated with drug action.

Dissociation between steroid receptor expression and cell proliferation in the human breast.

We have shown previously that estradiol stimulates cell proliferation and progesterone receptor (PgR) synthesis in luminal epithelial cells of the normal human breast. Approximately 10-15% of luminal epithelial cells within the normal breast express immunodetectable estrogen receptor (ER), but little is known about their distribution within lobules and their organization in relation to the smaller population of proliferating cells. Using normal human breast tissue, we show that ER-positive cells are distributed evenly throughout the mammary epithelium. Using double antibody immunofluorescence, we show that 96% of steroid receptor-positive cells synthesize both ER and PgR (n = 25). Double labeling with antibodies to either ER or PgR coupled with either [3H]thymidine histoautoradiography or with antibodies to the Ki67 proliferation antigen indicates that dividing cells are separate from those expressing the receptors (although they are often in close proximity). However, in contrast to the normal human breast, two-thirds of ER-positive human mammary tumors examined (n = 19) have a high proportion of dividing cells that are ER positive. These data are consistent with the hypothesis that cells in normal human breast epithelium are hierarchical in organization and support a model in which proliferation of ER-negative cells is controlled by paracrine factors released from ER-positive cells under the influence of estradiol. This organization may be disrupted in some tumors.

Effects of a pure antiestrogen on apoptosis and proliferation within human breast ductal carcinoma in situ.

Adjuvant antiestrogen (AE) therapy has been proposed for all women with ductal carcinoma in situ (DCIS). However, many cases of DCIS are of the high-grade, estrogen receptor (ER)-negative subtype that are unlikely to respond to AE treatment. Hormonal agents work by increasing apoptosis and/or decreasing cell proliferation; therefore, we studied the effect of a pure AE on levels of apoptosis and proliferation in human DCIS xenografts using an in vivo model. Women (n = 23) with mammographic microcalcification suggestive of DCIS were identified at the time of surgery (day 0), a sample of representative tissue was obtained, divided into multiple 2x2x1-mm xenografts, and implanted s.c. into female BALB/c nu/nu mice (eight xenografts/mouse). Day 0 grafts underwent immunohistochemical assessment of ER status. Fourteen days after implantation, four xenografts were retrieved and mice were randomly divided into one of three treatment groups: (a) insertion of a slow release 2-mg 17beta-estradiol pellet; (b) weekly 5-mg injections of the pure AE Faslodex (Zeneca Pharmaceuticals); and (c) injections of a control vehicle oil alone. After 2 weeks of treatment, the remaining four xenografts were retrieved from each mouse. Retrieved xenografts containing DCIS were assessed for morphological evidence of apoptotic cell death [apoptotic index (AI)] and cell proliferation (by immunohistochemical detection of the Ki67 proliferation antigen LI). Both AI and LI were higher in the day 0 specimens of 16 ER- DCIS lesions compared with 7 ER+ DCIS lesions (mean values, 1.47% versus 0.32% and 20.6% versus 3.1%; both P<0.0001). AI and LI values within ER- DCIS did not differ between xenografts exposed to 17beta-estradiol or AE treatment compared with the controls or pretreatment values (mean AI and LI in estradiol-treated, antiestrogen-treated, and control groups 1.04% versus 0.98% versus 1.29% and 17.2% versus 20.5% versus 17.7% respectively). In contrast, treatment of mice bearing ER+ DCIS xenografts with 17beta-estradiol raised both the AI (1.03% versus 0.40%, P = 0.03) and LI (11.0% versus 5.1%, P = 0.007) compared with controls. AE therapy of ER+ DCIS xenografts did not affect proliferation but resulted in higher apoptosis than in controls (0.9% versus 0.4% respectively, P = 0.04). AE therapy should be reserved for patients with estrogen receptor positive DCIS.

The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice.

Three h after whole-body irradiation (8 Gy) of C57BL x DBA/2 F1 mice, p53 protein was expressed strongly in the stem cell compartment of the small intestine but at lower levels in the colon. At this time, apoptotic cells were also observed in the stem cell position of the small intestine, with fewer in the colon. In mice without copies of the p53 gene (nulls), the levels of spontaneous apoptosis, in both the small intestine and the colon, were not different from wild-type. Irradiation of the nulls with 8 Gy of gamma-rays failed to induce any further apoptosis: the loss of p53 essentially rendered the epithelial cells, from both the small intestine and the colon, radioresistant. The response of the epithelial stem cells of the small intestine suggests that p53 may play a role in the deletion of damaged cells with carcinogenic potential, whereas this process is limited in the colon.

Expression of Id helix-loop-helix proteins in colorectal adenocarcinoma correlates with p53 expression and mitotic index.

Id helix-loop-helix (HLH) proteins function as regulators of cell growth and differentiation and when overexpressed can induce malignant transformation. In a series of 34 cases of primary human colorectal adenocarcinoma, immunoreactivity for Id1, Id2, and Id3 was found to be significantly elevated in tumor compared with normal mucosa (P = 0.001 for Id1 and Id2; P = 0.002 for Id3). No elevation of Id expression was observed in 17 cases of adenoma. Expression of Id1 and to a lesser extent of Id2 was correlated with mitotic index (P = 0.005 for Id1; P = 0.042 for Id2) in human adenocarcinomas, and expression of all three Id proteins was correlated with p53 immunoreactivity (a marker of mutational 'inactivation' of p53 function; P = 0.002 for Id1; P = 0.006 for Id2; P = 0.016 for Id3). In normal intestinal mucosa of p53-null mice and in spontaneous tumors arising in Min+/- mice, expression of all three Id proteins was also found to be up-regulated. Antisense oligonucleotide blockade of Id protein expression inhibited the proliferation of human adenocarcinoma cells. Enforced, ectopic expression of the E47 basic HLH (bHLH) protein in human adenocarcinoma cell lines efficiently sequestered endogenous Id proteins as Id-bHLH heterodimers, as shown by coimmunoprecipitation and subcellular colocalization studies. This led to growth arrest of the cells. Enforced overexpression of a mutant E47 protein, deficient in transactivation and DNA binding function, also partially inhibited cell growth. Taken together, these data imply that deregulated expression of Id proteins in colorectal adenocarcinoma arises at least in part as a consequence of loss of p53 function and contributes to the uncontrolled proliferation of tumor cells in colorectal cancer.

Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.

Apoptosis in small intestinal epithelial from p53-null mice: evidence for a delayed, p53-independent G2/M-associated cell death after gamma-irradiation.

The death of small intestinal epithelial cells has been characterized and quantitated after irradiation of mice rendered homozygously null for the p53 gene. In wild-type animals homozygous for p53 a rapid (4.5 h) elevation of p53 protein was observed in the proliferative compartment of the crypts after 8 Gy of irradiation. Cells underwent cell death by apoptosis in this region. We had reported previously a total repression of apoptosis in small intestinal crypt epithelia 4.5 h after the gamma-irradiation (8 Gy) of p53 homozygously null animals. Thus, while 400 apoptotic cells were observed in 200 half crypts taken from wild-type animals at 4.5 h, this fell to background levels (10-30) in the p53 null animals (Merritt et al., 1994) and did not increase by 12 h. However, we have now found a delayed initiation of a p53-independent apoptosis after 8 Gy of gamma-radiation: at 24 h, approximately 100 apoptotic cells were observed in 200 half crypts. This late wave of apoptosis was not observed after 1 Gy of gamma-radiation. The morphological appearance of this p53-independent apoptosis suggested that death may have arisen as the result of aberrant mitosis. Analysis of the regeneration of crypts 3 days after irradiation of mice with between 11 and 17 Gy showed that there was no significant increase (P=0.135) in the potential of clonogenic cells from the p53 null animals to repopulate the crypts. The data support the idea that a p53-independent apoptotic mechanism permits the engagement of apoptosis, probably by a mitotic catastrophe, after 8 Gy of gamma-irradiation in vivo and that a loss of p53 does not make these epithelial cells radioresistant in vivo to doses of 8 Gy and above. In contrast, irradiation with 1 Gy failed to induce a p53-independent apoptosis in vivo, suggesting that the p53 'sensor' of damage was more sensitive than that engaging the p53-independent mechanism of cell death.

Bcl-w is an important determinant of damage-induced apoptosis in epithelia of small and large intestine.

The potential role of the bcl-2 relative bcl-w as a physiological regulator of apoptosis in intestinal epithelia has been investigated. Immunoblots for bcl-w with new monoclonal antibodies revealed that it was expressed in the small intestine and colon, among other murine tissues, as well as in six human tumour cell lines of epithelial origin, including two colon carcinoma lines. To assess whether bcl-w regulates either spontaneous or damage-induced apoptosis in the small intestine or colon, apoptosis in intestinal crypts of bcl-w -/- and wild-type mice was quantified microscopically on a cell positional basis. Spontaneous apoptosis within crypt epithelia was not significantly increased by loss of bcl-w, in either the small intestine or midcolon. However, after treatment with the cytotoxic drug 5-fluorouracil or with gamma-radiation, the bcl-w-null animals exhibited substantially more apoptosis than their wild-type counterparts in both tissues. The greatest enhancement of apoptosis attributable to the absence of bcl-w (up to sixfold) occurred in the small intestine. Hence, bcl-w is an important determinant of damage-induced apoptosis in intestinal epithelia, and unlike bcl-2, which regulates only colonic apoptosis, plays a major role in small intestinal epithelium.

Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo.

The influence of bcl-2 and bax expression on apoptotic cell death in mouse intestinal epithelia was assessed using homozygously null mice. Apoptosis was induced in vivo by the enterotoxin 5-fluorouracil (5FU) or by gamma-irradiation and its cell positional incidence was assessed. 5FU and gamma-radiation treated bax-null mice surprisingly showed no reductions in apoptotic yield in the small intestine or midcolon at 4.5 h at cell positions in which both agents had previously been shown to strongly induce p53 protein expression. The colonic epithelia of 5FU treated bcl-2-null mice showed elevated levels of apoptosis at 4.5 h: from 48 apoptotic events in wild-type mice to 273 in the nulls, scoring 200 half crypts. The increase occurred specifically in the cell positions considered to harbour colonic stem cells, at the base of crypts, where there is selective expression of bcl-2. There was a modest but significant increase in apoptosis in the small intestine of the bcl-2-null mice although the epithelia of wild-type mice here are not immunohistochemically positive for bcl-2 protein. These findings show that bcl-2 plays a key role in determining the sensitivity of colonic stem cells to damage-induced death but that bax is not responsible for the p53-dependent induction of apoptosis in this context.

The importance of p53-independent apoptosis in the intestinal toxicity induced by raltitrexed (ZD1694, Tomudex): genetic differences between BALB/c and DBA/2 mice.

The thymidylate synthase inhibitor raltitrexed (ZD1694, Tomudex) induces greater intestinal toxicity, manifested as diarrhea and weight loss, in BALB/c than in DBA/2 mice. No convincing pharmacokinetic or pharmacodynamic reason for this strain difference has been established. We have investigated whether this strain difference in response to raltitrexed is related to differential susceptibilities of intestinal mucosae to undergo apoptosis and also whether p53 expression, a critical factor in 5-fluorouracil-induced intestinal apoptosis and toxicity, modulates this response. Ten mg/kg or 100 mg/kg raltitrexed were administered as single or double i.p. injections 24 h apart to BALB/c, DBA/2, and p53-/- mice. Apoptosis, mitosis, and tissue damage were assessed in intestinal epithelium, and animal weight was recorded. BALB/c mice developed diarrhea and weight loss following 100 mg/kg x2 raltitrexed, whereas DBA/2 mice did not. BALB/c mice were more sensitive than DBA/2 to induction of small-intestinal and colonic apoptosis 24 h following 100 mg/kg raltitrexed. Inhibition of mitosis was equivalent in both strains. Both strains showed histopathological damage to the small intestine after 100 mg/kg x2 raltitrexed, but only BALB/c mice demonstrated colonic damage. p53-null mice showed the same level of small intestinal apoptosis as their wild-type counterparts 24 h after 100 mg/kg x1 raltitrexed and also the same levels of intestinal toxicity 3, 5, and 7 days after 100 mg/kg x2 raltitrexed. Thus, BALB/c mice were more susceptible to induction of intestinal apoptosis by raltitrexed than DBA/2 mice and also demonstrated more histopathological damage in the colon correlating with the induction of diarrhea and weight loss. In contrast to 5-fluorouracil, the intestinal apoptosis and toxicity induced by raltitrexed were p53-independent.

Epidermal cell production rates.

Labeling and mitotic index data and estimates for the length of the S and M phases of the cell cycle from the literature have been compared with new data obtained over a 24-hr period from various epidermal sites in mouse. It has been found that values obtained at a single time of the day may give misleading results. All data have been interpreted in terms of cell production rates per epidermal proliferative unit (EPU). The final conclusions after consideration of cell production rates, labeling and mitotic indices, epidermal transit times, and epidermal structure are that dorsal and ear skin have rather similar cell production rates while tail and foot rates are 4 to 7 times higher. A comparison has also been made between the mouse results and the available human data. Hairless mouse dorsum appears from structural and proliferative aspects to be the best model for some regions of human skin. A new model has been proposed for epidermal proliferation based on the EPU. The model suggests a role for the Langerhans cells and suggests that there is a program of sequential cellular maturity in the EPU originating at the level of a central basal stem cell through committed proliferative cells and ending with cell loss from the skin surface.