Multifaceted role of hair follicle dermal cells in bioengineered skins.

BACKGROUND: The method of generating bioengineered skin constructs was pioneered several decades ago; nowadays these constructs are used regularly for the treatment of severe burns and nonhealing wounds. Commonly, these constructs are comprised of skin fibroblasts within a collagen scaffold, forming the skin dermis, and stratified keratinocytes overlying this, forming the skin epidermis. In the past decade there has been a surge of interest in bioengineered skins, with researchers seeking alternative cell sources, or scaffolds, from which constructs can be established, and for more biomimetic equivalents with skin appendages. OBJECTIVES: To evaluate whether human hair follicle dermal cells can act as an alternative cell source for engineering the dermal component of engineered skin constructs. METHODS: We established in vitro skin constructs by incorporating into the collagenous dermal compartment: (i) primary interfollicular dermal fibroblasts, (ii) hair follicle dermal papilla cells or (iii) hair follicle dermal sheath cells. In vivo skins were established by mixing dermal cells and keratinocytes in chambers on top of immunologically compromised mice. RESULTS: All fibroblast subtypes were capable of supporting growth of overlying epithelial cells, both in vitro and in vivo. However, we found hair follicle dermal sheath cells to be superior to fibroblasts in their capacity to influence the establishment of a basal lamina. CONCLUSIONS: Human hair follicle dermal cells can be readily interchanged with interfollicular fibroblasts and used as an alternative cell source for establishing the dermal component of engineered skin both in vitro and in vivo.

Analysis of the intra- and intertumoral heterogeneity of hypoxia in pancreatic cancer patients receiving the nitroimidazole tracer pimonidazole.

BACKGROUND: Hypoxia is thought to be an adverse feature of pancreatic cancer, but direct measurement in patients is technically challenging. To address this, we characterised the intra/interpatient heterogeneity of hypoxia in surgical specimens from patients who received the 2-nitroimidazole tracer pimonidazole pre-operatively. METHODS: Pimondazole was given intravenously 16-20 h before pancreatectomy, and the extent and intratumoral heterogeneity of hypoxia determined by image analysis applied to multiple tissue blocks stained by immunohistochemistry. Intra/interpatient heterogeneity was estimated by variance component analysis. RESULTS: Pimonidazole staining was analysed in 10 tumours. The extent of labelling varied amongst patients (0-26%), with a broader range of hypoxia in the epithelial (1-39%) compared with the stromal (1-13%) compartments. Variance component analysis demonstrated greater inter- than intrapatient variability of hypoxia, and that multiple (4-5) tumour sections are required to provide a consistent evaluation of its extent in individual tumours. CONCLUSIONS: There is significant intra- and intertumoral heterogeneity of hypoxia in pancreatic cancers, and these do not appear to be generally more hypoxic than other cancer types. This study establishes the feasibility to assess hypoxia in pancreatic cancer patients using pimonidazole, but questions the reliability of measurements made using a single tissue section.

Characterization of image quality and image-guidance performance of a preclinical microirradiator.

PURPOSE: To assess image quality and image-guidance capabilities of a cone-beam CT based small-animal image-guided irradiation unit (micro-IGRT). METHODS: A micro-IGRT system has been developed in collaboration with the authors' laboratory as a means to study the radiobiological effects of conformal radiation dose distributions in small animals. The system, the X-Rad 225Cx, consists of a 225 kVp x-ray tube and a flat-panel amorphous silicon detector mounted on a rotational C-arm gantry and is capable of both fluoroscopic x-ray and cone-beam CT imaging, as well as image-guided placement of the radiation beams. Image quality (voxel noise, modulation transfer, CT number accuracy, and geometric accuracy characteristics) was assessed using water cylinder and micro-CT test phantoms. Image guidance was tested by analyzing the dose delivered to radiochromic films fixed to BB's through the end-to-end process of imaging, targeting the center of the BB, and irradiation of the film/BB in order to compare the offset between the center of the field and the center of the BB. Image quality and geometric studies were repeated over a 5-7 month period to assess stability. RESULTS: CT numbers reported were found to be linear (R2 0.998) and the noise for images of homogeneous water phantom was 30 HU at imaging doses of approximately 1 cGy (to water). The presampled MTF at 50% and 10% reached 0.64 and 1.35 mm(-1), respectively. Targeting accuracy by means of film irradiations was shown to have a mean displacement error of [deltax, deltay, deltaz] = [-0.12, -0.05, -0.02] mm, with standard deviations of [0.02, 0.20, 0.17] mm. The system has proven to be stable over time, with both the image quality and image-guidance performance being reproducible for the duration of the studies. CONCLUSIONS: The micro-IGRT unit provides soft-tissue imaging of small-animal anatomy at acceptable imaging doses (< or =1 cGy). The geometric accuracy and targeting systems permit dose placement with submillimeter accuracy and precision. The system has proven itself to be stable over 2 yr of routine laboratory use (>1800 irradiations) and provides a platform for the exploration of targeted radiation effects in small-animal models.

Dynamics of micronuclei in rat skin fibroblasts after X irradiation.

In a previous study, we demonstrated DNA damage, expressed as micronuclei, in binucleate dermal fibroblasts obtained from human skin 2-9 weeks after fractionated radiotherapy. Here we assessed micronuclei in X-irradiated skin fibroblasts from 9-14-week-old female Lewis rats as a function of time after a single dose of radiation to determine the lifetime of such damage in the skin. After irradiation with 5, 10, 15 and 18 Gy, formation of micronuclei at 1 day or 2 months postirradiation increased up to about 10 Gy, with evidence for a plateau at higher doses. The time course of micronuclei present in the skin fibroblasts demonstrated a plateau region (approximately 20 days after 18 Gy and about 2 months after 10 Gy) before the number of micronuclei started to decline. Residual micronuclei were observed for more than 1 year after irradiation. Monomicronucleated cells predominated in fibroblasts from nonirradiated skin, whereas in fibroblasts from irradiated skin, multimicronucleated cells predominated and persisted (together with monomicronucleated cells) in the residual levels of damage at late times. The results suggest that DNA damage in dermal fibroblasts can be assayed by the micronucleus assay in samples from irradiated skin up to 1 month after irradiation for doses up to at least 10 Gy. Further studies are needed to define the dose-response relationship in detail.

Dynamic heterogeneity: rapid generation of metastatic variants in mouse B16 melanoma cells.

The ability of clonally derived lines of B16F1 and B16F10 melanoma cells to form experimental metastases in C57BL mice after intravenous injection was examined. Luria- Delbruck fluctuation analysis was applied to the results obtained with parallel subclones grown to small population sizes before testing for metastatic ability. The analysis demonstrated that variant cells capable of forming experimental metastases were generated in B16F1 cell populations at an effective rate of about 1.3 X 10(-5) per cell per generation while in B16F10 cell populations the effective rate of production was about 5 X 10(-5) per cell per generation. These results are consistent with a dynamic heterogeneity model of tumor progression. They suggest that the majority of cells in both lines are effectively nonmetastatic and that the higher metastatic ability of the B16F10 population may be due in part to a higher rate of generation of metastatic variants.

Enhanced metastatic dissemination to multiple organs by melanoma and lymphoma cells in timp-3-/- mice.

Identifying versatile inhibitors of metastasis that operate in multiple sites against distinct cancer cell types is important for designing novel therapeutics for metastasis. We show that multiple tissues of timp-3-/- mice are more susceptible to metastatic colonization. Overall, a 5-14-fold increase in liver and kidney colonization occurred by EL-4 lymphoma cells, and a twofold increase upon targeting B16F10 melanoma cells to the bone or lung of timp-3-/- mice. There was a general lack of macrophage or neutrophil localization to metastases in the liver, kidney and lung, and of osteoclasts to bone in both genotypes. Analysis of lung showed that proliferation or angiogenesis were unaltered within the metastatic colonies. Lung-trap assays revealed that initial tumor cell trapping was similar in the lung vasculature of timp-3-/- and wild-type mice. However, more tumor cells were found in timp-3-/- lungs at 48 and 96 h after tumor cell injection indicating more efficient extravasation and initial proliferation. Activation of pro-MMP-2 was greater in timp-3-/- lungs at these time points. These data demonstrate TIMP-3 functions to inhibit metastatic dissemination of diverse cancer cells to multiple organs. TIMP-3 regulates MMP-2 activation to limit tumor cell extravasation and subsequent colonization of the lung, without augmenting inflammatory cell response.

The p53-mediated G1 checkpoint is retained in tumorigenic rat embryo fibroblast clones transformed by the human papillomavirus type 16 E7 gene and EJ-ras.

Rat embryo fibroblast clones transformed with the human papillomavirus type 16 E7 gene and the H-ras oncogene (ER clones) fall into two groups on the basis of endogenous p53 genotype, wild type or mutant. We have compared these clones with the aim of indentifying physiological differences that could be attributed to p53 protein function. We show that all ER clones, regardless of p53 gene status, are tumorigenic and metastatic in severe combined immunodeficiency mice. We demonstrate that only the wild-type p53 protein expressed in ER clones is functional on the basis of its site-specific double-stranded DNA-binding activity and its ability to confer a G1 delay on cells following treatment with ionizing radiation. These data indicate that disruption of the p53 growth-regulatory pathway is not a prerequisite for the malignant conversion of rat embryo fibroblasts expressing the E7 gene and mutant ras. Differences in phenotype that were correlated with loss of p53 protein function included the following: serum-independent growth of ER clones in culture, decreased tumor doubling time in vivo, and increased radioresistance. In addition, we demonstrate the p53-dependent G1 checkpoint alone does not determine radiosensitivity.

Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts.

Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.

Melanocyte stimulating hormone peptides inhibit TNF-alpha signaling in human dermal fibroblast cells.

Alpha-melanocyte stimulating hormone (alpha-MSH) has been identified as a potent anti-inflammatory in various tissues including the skin. It has previously been shown in skin cell keratinocytes and melanocytes/melanoma cells that MSH peptides inhibit TNF-alpha stimulated NF-kappaB activity and intercellular adhesion molecule-1 (ICAM-1) upregulation. However, the precise anti-inflammatory role of MSH peptides in dermal fibroblasts is unclear. Some studies report on pro-inflammatory responses, while others on anti-inflammatory responses. The present study confirms MC1R expression in cultured human dermal fibroblasts and reports that the MSH peptides alpha-MSH and KP(-D-)V inhibit TNF-alpha stimulated NF-kappaB activity and ICAM-1 upregulation, consistent with an anti-inflammatory role. However, involvement of IkappaB-alpha regulation by either peptide was not confirmed, supporting a mechanism independent of the NF-kappaB inhibitor. In conclusion, alpha-MSH and KP(-D-)V peptides have an anti-inflammatory action on dermal fibroblast signaling by inhibiting the pro-inflammatory activity of TNF-alpha in vitro.

Macroprolactin(s): composition and reactivity in immunoassays and laboratory interpretation of results of an unusual patient serum.

BACKGROUND: Macroprolactin is a high molecular mass complex of prolactin that may be the cause of elevated serum prolactin as determined by immunoassay. The composition of macroprolactin and its reactivity in immunoassays are variable but the complex has minimal bioactivity in vivo. Hyperprolactinaemia due to unrecognized macroprolactinaemia can lead to misdiagnosis and mistreatment. METHODS: Serum from a patient with an unusual pattern of immunoreactivity was distributed to three users of each of the most popular immunoassays represented in the UK National External Quality Assessment Scheme (NEQAS) for prolactin. Clinical details were provided and participating centres were invited to investigate the prolactin content of the specimen according to their local protocol, and to comment on their results. The nature of the macroprolactin in the specimen was investigated in detail by gel filtration chromatography of the native serum and of the serum after adsorption of IgG with protein A, and by affinity chromatography with concanavalin A. RESULTS: Gel filtration studies revealed two peaks of macroprolactin in this serum. These macroprolactins were shown to be different in their IgG content and degree of glycosylation. All eight immunoassays reacted strongly with the macroprolactin present. The majority (78%) of centres that interpreted their results either demonstrated the presence of macroprolactin in the specimen, or suggested it as a likely cause of the hyperprolactinaemia. However, two centres inappropriately excluded macroprolactinaemia as the cause of the elevated prolactin, and a further two did not consider it at all. Data from previous UK NEQAS distributions (between 1996 and 2003) of macroprolactin containing sera are presented which suggest that the frequency of recognition of macroprolactin as a possible cause of hyperprolactinaemia has increased over time. CONCLUSIONS: Very high molecular mass forms of prolactin and the presence of multiple molecular mass forms, as detected in the case presented here, are uncommon. Also, the pattern of immunoreactivity reported in this specimen was unusual as most macroprolactins studied previously react less strongly in, for example, the Bayer ADVIA Centaur assay compared to the Roche E170 assay. Both peaks of macroprolactin in this serum reacted in all assays tested. This case highlights the variable nature and immunoreactive behaviour of macroprolactin species.

Effects of reoxygenation on cells from hypoxic regions of solid tumors: anticancer drug sensitivity and metastatic potential.

Tissue hypoxia in regions of solid tumors has been identified as a factor that may affect the behavior of cancer cells. In this study, cells were isolated from hypoxic regions of transplanted murine tumors and tested for sensitivity to anticancer drugs and ability to form experimental metastases. The tumors studied were KHT-C2-LP1 fibrosarcoma and SC-CVII squamous cell carcinoma in C3H mice and B16F10-A1 melanoma in C57BL mice. Our results indicate that the position of tumor cells relative to the vasculature, which determines the degree of tissue oxygenation, does not influence the in vitro sensitivity of cells to either doxorubicin or methotrexate. Conversely, 1-2 days after reoxygenation by introduction into culture, subpopulations of tumor cells demonstrated a transient increase in lung colonization ability. The most hypoxic cells exhibited a metastatic efficiency that was generally twice that of cells from well-oxygenated regions. This behavior is similar to behavior we observed in a previous study when tumor cells were exposed in vitro to conditions of extreme hypoxia. The findings in that study suggested that gene amplification associated with DNA over-replication is responsible for the enhanced metastatic potential, but we found no indication in the present study that gene amplification was involved in the effect observed with the hypoxic tumor subpopulations. These results provide additional evidence that reoxygenated cancer cells have a high colonization ability and that these cells may be important in the formation of distant metastases.

Further studies of the action of a partially purified bacteriocin against a murine fibrosarcoma.

We have reported previously that a partially purified bacteriocin (PPB) from Escherichia coli HSC10 is toxic to KHT cells growing in vivo as micrometastases but apparently has no activity against a tumor growing i.m. We report here experiments to investigate possible reasons for this difference. The PPB was shown to become less effective against micrometastases, initiated by i.v. injection of KHT cells, as the time between cell injection and PPB treatment increased. The kinetics of the loss of efficacy did not, however, correlate exactly with the growth kinetics of the nodules as assessed by survival following radiation treatment at different times after cell injection. This suggests the possibility of a diffusion limitation although it was found that s.c. injections of PPB were nearly as effective against micrometastases as i.p. injections. We also demonstrated that the lifetime of the majority of the toxic activity of PPB in vivo was relatively short (less than 1 day) and that the majority of its effect was not caused by stimulating macrophages to act against the tumor cells. The PPB was found to be cytotoxic to KHT cells in vitro but the effect was reduced at high cell density (approximately 10(6) cells/ml). The PPB did not induce an immune reaction against itself in C3H mice nor was it toxic to either bone marrow stem cells or jejunal crypt cells at doses which were effective against KHT micrometastases. We conclude that PPB may have potential as a cytotoxic agent to act against circulating tumor cells or very small deposits of tumor cells but is limited in its efficacy against larger tumor masses probably because of diffusional and/or cell density effects.

The response of hypoxic B16 melanoma cells to in vivo treatment with chemotherapeutic agents.

Survival curves are presented for the treatment of B16 melanomas with a range of single doses of cyclophosphamide (CY), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), or 1-(2-chloroethyl)-3-trans-4-methylcyclohexyl)-1-nitro-sourea (MeCCNU). When these four drugs are assessed in terms of the tumor cell kill at the lethal dose to 10% of the mice, MeCCNU is found to be much the most effective, followed by CCNU, and then CY and BCNU together. The superiority of MeCCNU is possibly related to the fact that it seems to be longer lived in the mice than are the other drugs. Combined drug and irradiation experiments have indicated that CY kills both oxygenated and hypoxic cells in the tumor, leaving proportions equal to those in the tumor prior to treatment, whereas BCNU preferentially spares the hypoxic cells. Since hypoxic cells constitute a population of cells that is at a distance from blood vessels, this result suggests that CY treatment of B16 melanomas is not limited by an inability of the drug to diffuse to cells away from blood vessels.

Effects of reoxygenation on cells from hypoxic regions of solid tumors: analysis of transplanted murine tumors for evidence of DNA overreplication.

Transient exposure of cultured cells to conditions of extreme hypoxia can induce DNA overreplication and the generation of cellular variants. This effect may be important for the development of tumor heterogeneity, since hypoxia may arise in solid tumors as a result of vascular insufficiency. We have investigated whether reoxygenation of cancer cells obtained from hypoxic regions of solid tumors is associated with DNA overreplication. Murine tumor cells were isolated from i.m. transplants as a function of their distance from the vasculature using a technique which involves in vivo staining of tumor tissues with the fluorochrome Hoechst 33342 (Chaplin et al., Br. J. Cancer, 51:569-572, 1985). Studies which determined the radiation sensitivity and cell cycle distribution of cells in the subpopulations indicated that cells were isolated from regions of the tumor which differed in oxygen levels. When KHT fibrosarcoma cells were isolated from hypoxic regions of tumors and introduced into culture (i.e., were reoxygenated), flow cytometric analysis revealed that they did not undergo any large scale DNA overreplication. These results indicate that hypoxic conditions which exist in transplanted tumors do not induce cells to undergo DNA overreplication to the same extent that is achieved after in vitro exposure of cells to hypoxia. We also found that at high concentrations (10 microM) the Hoechst dye itself induced DNA overreplication. These concentrations are frequently used to vitally stain cells for sorting according to DNA content, and this effect must be considered in the interpretation of such experiments.

Activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide against Chinese hamster cells in multicellular spheroids.

The cytotoxic activity of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), a novel acridine derivative with clinical antitumor activity, has been examined in multicellular spheroids grown from Chinese hamster V79-171b cells. m-AMSA is much less effective against cells within these tumor-like structures than it is against exponential-phase V79-171b cells in monolayer cultures, the initial D0 of the survival curve for the latter being approximately 10-fold lower than for the former following a 60-min exposure to the drug. The resistance of spheroid cells to m-AMSA appears to be at least partially a result of the noncycling or slowly cycling state of the majority of these cells, although they are more sensitive than cells in plateau-phase monolayers. A further component of resistance in spheroids requires the presence of an intact spheroid structure and may be due to drug transport limitations. The use of sequential trypsinization techniques to recover cells at varying depths within spheroids demonstrates that a 60-min m-AMSA treatment preferentially kills cells nearest the spheroid surface, suggesting that tumor cells at a distance from the vasculature may limit the efficacy of m-AMSA in vivo.

Tumor heterogeneity and stability of the metastatic phenotype of mouse KHT sarcoma cells.

Heterogeneity in metastatic ability has been demonstrated in model systems for in vitro-cloned cell lines for a number of different tumors. We have examined the clonal diversity of mouse KHT sarcoma cells cloned either in vitro or in vivo by determining their ability to form lung colonies following i.v. injection into syngeneic mice. A wide range of metastatic ability was found in both the in vitro- and in vivo-isolated clones, suggesting that the diversity observed is not due to any selection occurring during in vitro growth. The stability of four in vitro-isolated clones, two of high metastatic and two of low metastatic ability, was then studied over a period of 3 to 4 months of growth in vitro. The phenotype of the highly metastatic cells remained relatively stable, declining only slightly over time. The clones with low metastatic ability, however, demonstrated a significant increase in ability to form lung colonies over the first 30 days in culture before becoming stable at levels approximately 10-fold higher than their original values. Even after this increase, however, there remained a difference of about a factor of 10 in the metastatic ability of the high and low pairs of clones. It was found that the number of lung colonies formed by all four cell lines was significantly increased when plastic microspheres were injected with the cell suspension. The cells with low metastatic ability were affected to a greater degree by the microspheres, resulting in the elimination of the difference between the four clones after 30 days in culture. This result suggests that the use of microspheres may provide a means to distinguish different cellular properties which affect the ability of cells to form lung metastases.

Acute (cyclic) hypoxia enhances spontaneous metastasis of KHT murine tumors.

Hypoxia exists in most human and rodent solid tumors and has been shown to correlate with poor survival in carcinoma of the cervix, carcinoma of the head and neck, and soft tissue sarcoma. It exists both chronically, due to the poorly organized vasculature of solid tumors, and acutely, due to fluctuations in blood flow. It has been found that tumors that are more hypoxic are more likely to metastasize in humans and in rodent models, and it has been demonstrated that exposure of tumor cells to hypoxia in vitro can transiently enhance their metastatic potential when they are reinjected i.v. into mice. The purpose of the present study was to determine whether experimentally imposed hypoxic stress in vivo, either chronic or acute, affects the process of spontaneous metastasis in tumor-bearing mice. We exposed mice bearing KHT tumors to low oxygen conditions (5-7% O(2) breathing) daily during tumor growth in an attempt to induce additional chronic (2 h/day) and acute (12 x 10 min/day) hypoxia in their tumors. By monitoring tumor pO(2) levels over the course of treatment, we demonstrated that these treatments produce acute and chronic hypoxia within the tumor tissue. The acute but not the chronic hypoxia treatment significantly increased the number of spontaneous microscopic lung metastases in the mice by a factor of about 2, and the results suggest that this effect was due to the changes induced in the primary tumor. This study describes a novel method for studying the effects of hypoxia in solid tumors and demonstrates that acute and chronic hypoxia can have different effects on tumor cell behavior in vivo.

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines.

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.

Toxicity of 4'-(9-acridinylamino)methanesulfon-m-anisidide in exponential- and plateau-phase Chinese hamster cell cultures.

The antitumor acridine derivative 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) is at present being evaluated in Phase 2 clinical trials. Exposure of exponential-phase Chinese hamster V79-171b cells to physiologically attainable concentrations of m-AMSA for 60 min generates survival curves with little or no threshold region and an initial D0 of 0.245 +/- 0.019 (S.D.) microM under standard conditions of assay. A minor subpopulation of apparently drug-resistant cells is revealed at low survivals, but these cells on culturing do not display a stable drug-resistant phenotype. m-AMSA survival curves for Chinese hamster ovary cells display features similar to the above. Sensitivity of V79-171b cells to m-AMSA is maximal near pH 7.2 and is markedly reduced by the presence of fetal calf serum. Hypoxia has little effect on the toxicity of m-AMSA, and repair of potentially lethal damage has not been observed after treatment with this agent. Noncycling plateau-phase V79-171b or Chinese hamster ovary cells are markedly less sensitive to m-AMSA than are early log-phase cells. This resistance to m-AMSA appears to be related to the slowly cycling or noncycling status of plateau-phase cells, suggesting that the cytokinetic character of cell populations in vivo will be a major determinant of their sensitivity to this drug. However, the increase in resistance to m-AMSA during growth into plateau-phase appears to commence well before departure from exponential growth can be detected and may thus be a consequence of metabolic changes more subtle than the transition from a cycling to a noncycling state.

Mutator phenotype in Msh2-deficient murine embryonic fibroblasts.

Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA mismatch repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse embryo fibroblasts with E7 and Ras oncogenes or mutant p53. Spontaneously immortalized cells derived from the primary cultures were also studied. To determine whether these cells developed a mutator phenotype similar to that found in colon cancer cells deficient in mismatch repair, we measured mutation rates, microsatellite instability, and sensitivities to a range of DNA-damaging agents. The mutator phenotype detected in the E7 and Ras or mutant p53-immortalized Msh2-/- mouse cells was similar to that found in human mismatch repair-deficient colorectal carcinoma cell lines. Mutation rates to ouabain resistance were increased 8-12-fold relative to lines from Msh2+/+ mice, and microsatellite instability was detectable in 12-18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ line. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine relative to Msh2+/+ cells. In contrast, these lines showed various responses to UV light and cis-platinum, suggesting that mismatch repair deficiency was not the sole determinant for sensitivity to these DNA-damaging agents. Particular attention was paid to the properties of cells heterozygous for the Msh2 mutant gene, which would mimic the situation of an HNPCC carrier. However, our studies failed to reveal any properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observation is consistent with the model that inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis in HNPCC patients.