atm and p53 cooperate in apoptosis and suppression of tumorigenesis, but not in resistance to acute radiation toxicity.

Mutations in atm and p53 cause the human cancer-associated diseases ataxia-telangiectasia and Li-Fraumeni syndrome, respectively. The two genes are believed to interact in a number of pathways, including regulation of DNA damage-induced cell-cycle checkpoints, apoptosis and radiation sensitivity, and cellular proliferation. Atm-null mice, as well as those null for p53, develop mainly T-cell lymphomas, supporting the view that these genes have similar roles in thymocyte development. To study the interactions of these two genes on an organismal level, we bred mice heterozygous for null alleles of both atm and p53 to produce all genotypic combinations. Mice doubly null for atm and p53 exhibited a dramatic acceleration of tumour formation relative to singly null mice, indicating that both genes collaborate in a significant manner to prevent tumorigenesis. With respect to their roles in apoptosis, loss of atm rendered thymocytes only partly resistant to irradiation-induced apoptosis, whereas additional loss of p53 engendered complete resistance. This implies that the irradiation-induced atm and p53 apoptotic pathways are not completely congruent. Finally-and in contrast to prior predictions-atm and p53 do not appear to interact in acute radiation toxicity, suggesting a separate atm effector pathway for this DNA damage response and having implications for the prognosis and treatment of human tumours.

Idiotype-like molecules on cells of a human T cell leukemia.

Two monoclonal antibodies were obtained that showed unique specificities for the leukemic T cells used for immunization. One antibody, S160, was totally specific for the antigen. The other antibody, S511, also reacted with a small population of normal T cells. This was made especially evident by concentrating these normal T cells with the antibody. Considerable evidence was obtained that both antibodies reacted with the same membrane molecules. In the unreduced state a major component of approximately 80 kdaltons was observed; after reduction this split into two components of approximately 43 and approximately 38 kdaltons. The reaction of the two antibodies with different antigenic sites on the same molecule, one representing a private site and the other a more cross-reactive site, strongly suggests an antibodylike molecule, but composed of polypeptide chains differing from immunoglobulins.

Age-resolving osteopetrosis: a rat model implicating microphthalmia and the related transcription factor TFE3.

Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.

T cell antiidiotypic antibodies reveal differences between two human leukemias.

Two different human T cell leukemias were compared, using antiidiotype-like murine monoclonal antibodies. In each case these antibodies immunoprecipitated disulfide-linked heterodimer molecules from their respective leukemic cells. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the two idiotype-bearing molecules a major difference in molecular weight was observed, which could be attributed to a similar difference in size of the heavily iodinated chain of either heterodimer. The lightly iodinated chains of both molecules co-migrated at 43 Kd, but appeared to have different isoelectric points on two-dimensional gel analysis. The possibility that these two different heterodimers correspond to different classes of the putative T cell receptor for antigen is discussed. Assays of proliferation of the leukemic cells using Sepharose-bound antiidiotype-like monoclonal antibody showed that one of the leukemic cell types proliferated readily in response to its antiidiotypic antibody. This proliferation was not associated with measurable production of IL-2 and appeared to be a direct effect of the antiidiotypic antibody, which may mimic antigen in its interaction with the T cell receptor for antigen. The other leukemic cell type did not respond to Sepharose-bound antiidiotypic antibody and was generally unresponsive to lymphokines and mitogens. It is possible that the two leukemic cell types represent different stages of T cell differentiation.

Melanocyte photobiology, ultraviolet radiation and melanoma.

Of all the organs of the human body, the skin is most commonly affected by malignancy, and ultraviolet radiation has long been implicated as the primary mutagenic exposure leading to the development of many cutaneous cancers. However, as research in this field has continued, it has become clear that the effect of ultraviolet radiation on the skin is quite complex. Distinct cell types within the skin exhibit differing responses to ultraviolet radiation, and even within the same cell type, divergent effects may be observed; depending on the dose or wavelength of the radiation, or the maturational state of the affected cell, unique responses can be elicited. Melanocytes form a minor component of the outermost layer of skin, but they have an enormous impact on not only the appearance of the skin, but also the ability of the skin to withstand exposure to ultraviolet radiation. In addition, melanocytes give rise to melanoma, one of the most deadly types of skin cancer. Clearly, it is critical that we achieve a better understanding of the effect of ultraviolet radiation on melanocytes, and that we clarify its role in the oncogenesis of melanoma. Although the picture is far from complete, the mechanisms by which melanocytes respond to ultraviolet radiation are beginning to be elucidated, and, as these pathways emerge, they offer new targets for chemopreventive and chemotherapeutic intervention.

Loss of matrix adhesion triggers rapid transformation-selective apoptosis in fibroblasts.

Cell-matrix and cell-cell adhesion are recognized physiological determinants of cell growth and survival. In epithelial and endothelial cell systems, oncogenic transformation has in several cases been shown to confer resistance to apoptosis upon depriving cells of substrate adhesion. We examined the effects of oncogenic transformation in adherent versus adhesion- deprived primary embryonic fibroblasts. Whereas untransformed early passage fibroblasts undergo cell cycle arrest, their Myc/Ras- or E1A/Ras-transformed counterparts rapidly enter apoptosis when placed into suspension. This phenomenon also occurs upon incubation with a soluble, RGD-containing integrin ligand and is blocked by a peptide antagonist to ICE family proteases or by aggregation of cells plated at high density. Loss of wild-type p53 modulates the kinetics but does not abrogate this death pathway. Transformation with activated Src rather than Ras rendered fibroblasts selectively resistant to adhesion-dependent apoptosis, an effect likely related to Src's role in integrin signaling, while simultaneously sensitizing the cells to radiation-induced apoptosis. Thus cell adhesion events regulate transformation-selective apoptosis in fibroblasts and provide potentially important targets for understanding and interfering with tumor cell viability.

Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes.

Melanocyte differentiation characterized by an increased melanogenesis, is stimulated by alpha-melanocyte-stimulating hormone through activation of the cAMP pathway. During this process, the expression of tyrosinase, the enzyme that controls melanin synthesis is upregulated. We previously showed that cAMP regulates transcription of the tyrosinase gene through a CATGTG motif that binds microphthalmia a transcription factor involved in melanocyte survival. Further, microphthalmia stimulates the transcriptional activity of the tyrosinase promoter and cAMP increases the binding of microphthalmia to the CATGTG motif. These observations led us to hypothesize that microphthalmia mediates the effect of cAMP on the expression of tyrosinase. The present study was designed to elucidate the mechanism by which cAMP regulates microphthalmia function and to prove our former hypothesis, suggesting that microphthalmia is a key component in cAMP-induced melanogenesis. First, we showed that cAMP upregulates the transcription of microphthalmia gene through a classical cAMP response element that is functional only in melanocytes. Then, using a dominant-negative mutant of microphthalmia, we demonstrated that microphthalmia is required for the cAMP effect on tyrosinase promoter. These findings disclose the mechanism by which cAMP stimulates tyrosinase expression and melanogenesis and emphasize the critical role of microphthalmia as signal transducer in cAMP-induced melanogenesis and pigment cell differentiation.

Fission track age of magnetic anomaly 10: a new point on the sea-floor spreading curve.

A portion of basaltic glass retrieved from an abyssal hill in the northeast Pacific has been dated by the fission track method. The sample location corresponds to magnetic anomaly 10 believed to have resulted from sea-floor spreading. The age of this sample is 35 +/- 5 million years, which is in agreement with the previously proposed age of 31 to 32 million years based on linear extrapolation of measured recent spreading rates. This observation upholds the suggestions of other authors on the time variation of sea-floor spreading for the last 30 million years in various parts of the world ocean basin.

Potassium: argon dating of iron meteorites.

The potassium:argon age of the metal phase of Weekeroo Station iron meteorite, determined by neutronactivation analysis, is about 10(10) years; it is similar to ages previously measured for other iron meteorites, but distinctly disagrees with a strontium: rubidium age of 4.7 X 10(9) years measured by other workers on silicate inclusions in this meteorite.

Ages of pacific deep-sea basalts, and spreading of the sea floor.

Potassium-argon determinations of age from whole-rock samples of tholeiitic basalts, dredged from the crest of the East Pacific Rise and from the flanks of three seamounts at varying distances from the crest, show that the crest is younger than 1 million years and that age does not correlate with distance from the crest. Our data, however, do not necessarily oppose the general concept of spreading of the ocean floor.

p53 status and the efficacy of cancer therapy in vivo.

The therapeutic responsiveness of genetically defined tumors expressing or devoid of the p53 tumor suppressor gene was compared in immunocompromised mice. Tumors expressing the p53 gene contained a high proportion of apoptotic cells and typically regressed after treatment with gamma radiation or adriamycin. In contrast, p53-deficient tumors treated with the same regimens continued to enlarge and contained few apoptotic cells. Acquired mutations in p53 were associated with both treatment resistance and relapse in p53-expressing tumors. These results establish that defects in apoptosis, here caused by the inactivation of p53, can produce treatment-resistant tumors and suggest that p53 status may be an important determinant of tumor response to therapy.

Autoimmune sera reactive with Sm antigen contain high levels of RNP-like antibodies.

Ribonucleoprotein particles containing Sm antigen were separated from particles containing both Sm and RNP antigens by ion-exchange chromatography to study the recognition of these antigens by autoimmune sera. By using the separated antigens, anti-Sm and/or anti-RNP antibodies were detected in approximately 60% of sera from systemic lupus erythematosus patients by both enzyme-linked immunosorbent assay and immunoprecipitation of radiolabeled antigens followed by analysis on sodium dodecyl sulfate-polyacrylamide gels. These antibodies were detected in 30% of the same sera using the standard passive hemagglutination technique. Competition experiments demonstrated that all of the sera tested that contained anti-Sm antibodies also had anti-RNP-like reactivity. This latter reactivity usually represented 80% or more of the total Sm and RNP binding activity in lupus sera. The binding to RNP-like determinants by several of the sera was uniquely resistant to treatment of the antigen with snake venom exonuclease. These studies indicate that humoral immunity against Sm and RNP antigens in systemic lupus erythematosus is directed primarily against a single type of ribonucleoprotein particle in which the two antigens are physically associated. The specific binding to a single type of ribonucleoprotein particle suggests that this particle may be especially immunogenic and that it might play an important role in induction of the humoral immune response to Sm and RNP.

Regulation of proliferation-survival decisions during tumor cell hypoxia.

Hypoxia may influence tumor biology in paradoxically opposing ways: it is lethal as a direct stress trigger, yet hypoxic zones in solid tumors harbor viable cells which are particularly resistant to treatment and contribute importantly to disease relapse. To examine mechanisms underlying growth-survival decisions during hypoxia, we have compared genetically related transformed and untransformed fibroblast cells in vitro for proliferation, survival, clonogenicity, cell cycle, and p53 expression. Hypoxia induces G0/G1 arrest in primary fibroblasts but triggers apoptosis in oncogene-transformed derivatives. Unexpectedly, the mechanism of apoptosis is seen to require accumulated acidosis and is rescued by enhanced buffering. The direct effect of hypoxia under nonacidotic conditions is unique to transformed cells in that they override the hypoxic G0/G1 arrest of primary cells. Moreover, when uncoupled from acidosis, hypoxia enhances tumor cell viability and clonogenicity relative to normoxia. p53 is correspondingly upregulated in response to hypoxia-induced acidosis but downregulated during hypoxia without acidosis. Hypoxia may thus produce both treatment resistance and a growth advantage. Given strong evidence that hypoxic regions in solid tumors are often nonacidotic (G. Helmlinger, F. Yuan, M. Dellian, and R. K. Jain, Nat. Med. 3:177-182, 1997), this behavior may influence relapse and implicates such cells as potentially important therapeutic targets.

Risk of melanocytic nevi and nonmelanoma skin cancer in children after allogeneic hematopoietic stem cell transplantation.

There is a known increased risk of skin cancer in the adult population after hematopoietic stem cell transplantation (HSCT). However, late dermatologic effects that children may experience after HSCT have not been well described. The primary objective of this study was to characterize nevi and skin cancers affecting children after allogeneic HSCT. A cross-sectional cohort study of 85 pediatric HSCT recipients and 85 controls matched for age, sex and skin phototype was performed at a single institution. All participants underwent a full skin examination. Median age at study visit was 13.8 years in HSCT patients with median time post-HSCT of 3.6 years. HSCT patients had significantly more nevi than control patients (median (range): 44 (0-150) vs 11 (0-94), P<0.0001). HSCT patients also had significantly more nevi >5 mm in diameter and atypical nevi than controls. Factors associated with increased nevus count included malignant indication for HSCT, pretransplant chemotherapy, TBI exposure and myeloablative conditioning. A total of 16.5% of HSCT patients developed cancerous, precancerous lesions and/or lentigines. Our study suggests that pediatric HSCT recipients have an increased risk of benign and atypical melanocytic proliferations and nonmelanoma skin cancer that can manifest even during childhood.

A CD44 survival pathway triggers chemoresistance via lyn kinase and phosphoinositide 3-kinase/Akt in colon carcinoma cells.

A major obstacle to successful treatment of colorectal cancer is chemotherapy resistance. Enhanced expression of variant CD44 isoforms has been associated with aggressive tumor behavior, prompting the question of whether signaling from this receptor might modulate drug sensitivity. Activation of variant CD44 in colon carcinoma cell lines triggered resistance to the drug 1,3-bis(2-chloroethyl)-1-nitrosurea. Resistance was induced by monoclonal antibodies directed against epitopes independent of the hyaluronate-binding region but was not triggered by identical treatment of a carcinoma line expressing the standard CD44 isoform. We observed that variant CD44 produced activation of the src-family tyrosine kinase lyn. Moreover, overexpression of dominant-active lyn recapitulated chemoresistance via a pathway shown to involve activation of phosphoinositide 3-kinase and Akt. These results establish a novel role for CD44 in determining survival of colon carcinoma cells through lyn kinase and Akt. The ability to suppress apoptosis might play a critical role in the onset and development of colorectal malignancies.

Genetic interactions between atm and p53 influence cellular proliferation and irradiation-induced cell cycle checkpoints.

Ataxia-telangiectasia and Li-Fraumeni syndrome, pleiotropic disorders caused by mutations in the genes atm and p53, share a marked increase in cancer rates. A number of studies have argued for an interaction between these two genes (for comprehensive reviews, see M. S. Meyn, Cancer Res., 55: 5991-6001, 1995, and M. F. Lavin and Y. Shiloh, Annu. Rev., Immunol., 15: 177-202, 1996). Specifically, atm is placed upstream of p53 in mediating G1-S cell cycle checkpoint control, and both atm and p53 are believed to influence cellular proliferation. To analyze the genetic interactions of atm and p53, mouse embryonic fibroblasts (MEFs) homozygously deficient for both atm and p53 were used to assess cell cycle and growth control. These double-null fibroblasts proliferate rapidly and fail to exhibit the premature growth arrest seen with atm-null MEFs. MEFs null for both atm and p53 do not express any p21(cipl/wafl), showing that p53 is required for p21(cipl/wafl) expression in an atm-null background. By contrast, homozygous loss of either atm, p53, or both results in similar abnormalities of the irradiation-induced G1-S cell cycle checkpoint. Our results suggest two separate pathways of interaction between atm and p53, one linear, involving G1-S cell cycle control, and another more complex, involving aspects of growth regulation.

DNA bending and the curious case of Fos/Jun.

DNA bending has been implicated as an important regulatory mechanism in several processes involving protein-DNA interactions. Various methods for examining intrinsic and protein-induced DNA bending may lead to different conclusions. For the Fos and Jun transcription factors, this has resulted in controversy over whether these factors significantly bend DNA at all.

Mechanisms of apoptotic cell death.

Apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. Several mediators of programmed cell death have been identified and signals of apoptosis have been found to utilize common pathways, some of which have been elucidated. This review focuses on a number of apoptotic systems that have been widely studied and discuss recent progress and opinion in these areas. These include studies on Fas signaling, tumor suppressor genes, cell cycle interfaces, stress responses, genetic systems, and the Bcl-2 family. Understanding apoptosis from these perspectives sheds substantial light on processes of biological homeostasis. Furthermore, the ability to manipulate the apoptotic response may lead to novel therapeutic interventions in cancer and other diseases.

Modulation of microphthalmia-associated transcription factor gene expression alters skin pigmentation.

The microphthalmia-associated transcription factor is implicated in melanocyte development and in the regulation of melanogenesis. Microphthalmia-associated transcription factor is thought to bind to the M-box promoter elements of tyrosinase, tyrosinase-related protein-1 and dopachrome tautomerase/tyrosinase-related protein-2 and transactivate these genes, resulting in increased pigmentation. Using a luciferase reporter construct driven by the microphthalmia-associated transcription factor promoter, we identified agents that modulate microphthalmia-associated transcription factor promoter activity. Changes in endogenous microphthalmia-associated transcription factor expression levels upon treatment with these agents were confirmed using northern and western blots, and their pigmentary modulating activities were demonstrated. Ultraviolet B irradiation and traditional Chinese medicine-1, a natural extract used in traditional Chinese medicine, upregulated microphthalmia-associated transcription factor gene expression and enhanced tyrosinase activity in vitro. Dihydrolipoic acid, lipoic acid, and resveratrol reduced microphthalmia-associated transcription factor and tyrosinase promoter activities. These agents also inhibited the forskolin- and ultraviolet B-stimulated promoter activities of these genes and significantly reduced tyrosinase activity in melanocyte cultures, resulting in depigmentation. Overexpressed microphthalmia-associated transcription factor was capable of rescuing the repressive effects of these compounds on the cotransfected tyrosinase promoter. Dark-skinned Yucatan swine treated with these agents showed visible skin lightening, which was confirmed histologically, whereas ultraviolet B-induced tanning of light-skinned swine was inhibited using these agents. Our findings suggest that modulation of microphthalmia-associated transcription factor expression can alter skin pigmentation and further confirm the central role of microphthalmia-associated transcription factor in melanogenesis.

Apoptosis in tumorigenesis and cancer therapy.

Apoptosis is a morphologically and biochemically distinct form of cell death which can be triggered by a variety of extracellular agents during both normal development as well as in adult pathological states. Much progress has recently been made in understanding the molecular pathways which regulate this process as well as new intersections between these. A direct interaction between components of the 'executioner'--the ICE-family of cysteine proteases--and the Bcl-2 family of proteins, which modulate a cell's propensity to undergo apoptosis, has recently been demonstrated. New pathways to cell survival, like the PI3-K/Akt signal transduction pathway, are also providing new clues as to the regulation of cell death by growth factors and extracellular matrix for example. The links which exist between apoptosis and cancer research are several. Genetic alterations in components of the apoptosis pathway occur during tumorigenesis and confer resistance to a variety of physiological (oncogene-induced cell death, loss of adhesion, growth under hypoxia) as well as therapeutic (chemotherapy and radiation) death triggers. Similarly, antineoplastic therapies are thought to induce tumor cell apoptosis, and consequently, common mutations in apoptosis-regulatory genes carry a poor prognosis for the patient. A more detailed understanding of the biochemistry of apoptosis and the ways in which it is disabled in tumors will likely reveal new transformation selective death triggers which stimulate cell death in ways independent of components like p53 and increase the therapeutic window of these drugs in the clinics.