Chédiak-Higashi gene in humans I. Impairment of natural-killer function.

Natural-killer (NK)-cell function was profoundly depressed in donors homozygous for the Chediak-Steinbrinck-Higashi syndrome (C-HS) gene when compared with age- and sex-matched normals. This apparent defect was not simply a result of a delayed response because little cytolysis was evident in kinetics experiments even after 24 h of incubation. NK cells from C-HS donors failed to lyse adherent (MDA, CEM, and Alab) or nonadherent (K562 and Molt-4) tumor cell lines or nontransformed human fetal fibroblasts. Therefore, the apparent C-HS defect was not a result of a shift in target selectivities. In addition, the depressed reactivity did not appear to be a result of suppressor cells or factors because: (a) enriched NK populations (nonadherent lymphocytes bearing receptors for the Fc portion of IgG) from C-HS donors were low in NK-cell function, (b) C-HS mononuclear cells did not inhibit the cytotoxicity of normal cells in coculture experiments, and (c) cells from the C-HS donors remained poorly reactive even after culture for up to 7 d. The nature of the defective NK activity in C-HS patients is not clear but may lie within the lytic mechanism rather than at the level of the recognition structure or population size because the frequency of target-binding cells was normal. In vitro NK activity could be partially restored by interferon treatment. Combined with the results presented in the following paper (4), these observations suggest that the C-HS gene causes a selective immunodeficiency disorder, mainly involving NK cells. This finding, in conjunction with the high incidence of spontaneous possibly malignant, lymphoproliferative disorders in these patients, may have important implications regarding the theory of immune surveillance mediated by NK cells.

Chédiak-Higashi gene in humans. II. The selectivity of the defect in natural-killer and antibody-dependent cell-mediated cytotoxicity function.

Antibody-dependent cell-mediated cytolysis (ADCC) of human tumor cells by FcR(+) nonadherent effector lymphocytes as well as natural killer (NK) activity was markedly impaired in Chediak-Steinbrinck-Higashi Syndrome (C-HS) patients. Compared to a more than 400-fold defect in NK activity in terms of lytic units, the abnormal ADCC response in C-HS donors was 24-fold below normal suggesting a partial but not complete overlap of lymphocytes or lytic mechanisms responsible for ADCC and NK. The ADCC mechanism against erythrocyte targets, however, was normal, thereby suggesting a qualitative difference in these two forms of ADCC. Other effector-cell functions against tumor-cell targets were normal as measured by (a) spontaneous cytolysis mediated by monocytes, (b) spontaneous cytostasis mediated by neutrophils, and (c) lectin-dependent cytolysis mediated by neutrophils. Although one C-HS patient was low in lectin-dependent cytolysis mediated by lymphocytes, the other C-HS patient was normal, thereby suggesting that cytolytic T function was not linked to the NK-ADCC defect. In addition, the proliferative response to T-dependent mitogens was also relatively normal. These results, combined with other studies showing normal cell-mediated and humoral immunity in these same patients, suggest that patients with C-HS have an immunodeficiency which is selective for NK and ADCC activity.

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.

Enhanced natural killer sensitivity with concomitant clonal selection for cells bearing homogeneously staining regions in the human melanoma cell line MeWo upon induction of differentiation with theophylline.

Culture of the human melanoma cell line MeWo in the presence of 1 mM theophylline was associated with an increase in susceptibility to natural killer (NK)-mediated cytolysis. The phenomenon was detected as early as 72 hours after initiation of theophylline treatment, reaching maximum values at 3-4 weeks and remaining stable for longer than 3 months of testing, provided the cells were maintained in the presence of theophylline. The alteration in target sensitivity was selective for NK-mediated cytolysis, since other mechanisms of cell-mediated cytolysis, including antibody-dependent cell-mediated cytotoxicity and monocyte-mediated and lectin-induced cytolysis, were comparable between untreated and treated cells. The enhanced susceptibility of theophylline-treated cultures to NK lysis, as compared to NK lysis susceptibility of untreated MeWo cells, was not significantly changed by pretreatment of effector lymphocytes with interferon. Evidence for differentiation in theophylline-treated cultures was obtained. In addition, however, cytofluorometric and karyologic analysis revealed the existence of two subpopulations of differing ploidy in the MeWo line. The hypodiploid, NK-sensitive subpopulation, bearing homogeneously staining regions on two chromosomes, could be selected by growth in theophylline. Therefore, selection of subpopulations in heterogeneous tumor cell lines by chemical inducers suggests an alternative and novel mechanism for enhancement of NK sensitivity.

Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma cell line.

The human melanoma cell line MeWo was found to contain two populations of cells, one containing 83 chromosomes (hypotetraploid) and the other containing 43 chromosomes (hypodiploid). All of the hypodiploid cells, but none of the hypotetraploid cells, contained chromosomes with long homogeneously staining regions (HSR's) when examined with quinacrine fluorescence. These long HSR's on an X-chromosome and derivative chromosome 15, produced characteristic patterns of positive- and negative-staining areas along the HSR's with both conventional Giemsa (G) staining and C-banding. The C- and G-positive regions stained with distamycin A-DAPI, which is specific for the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and Y. DNA extracted from MeWo cells and digested with the restriction enzymes KpnL or Sau3A exhibited marked amplification of a 1.8-kilobase fragment. The amplified Sau3A fragment (D15Z1) was cloned, mapped, and partially sequenced. The sequenced region contained a five-base-pair repeat unit (adenine-adenine-thymine-guanine-guanine) that has undergone considerable divergence. Estimates of the size of the HSR's and the amount of amplified DNA suggested that each HSR contained at least 30,000 copies of the 1.8-kb KpnL fragment, representing about 50% of each HSR. The amplified sequence was identified as one member of the previously described KpnL family of repeated sequences. In situ hybridization of D15Z1 to MeWo metaphase chromosomes resulted in heavy labeling over both HSR's. These data suggested that centromeric heterochromatin and neighboring sequences on chromosome 15 have been amplified and some of this material translocated to the X-chromosome.

Nerve growth factor receptors of human tumors of neural crest origin: characterization of binding site heterogeneity and alteration by theophylline.

Heterogeneous populations of saturable specific high-affinity binding sites for the biologically active subunit of the 7S nerve growth factor complex purified from mouse submaxillary gland (NGF) are detected on human tumor cells of neural crest origin. Detailed studies of the melanoma cell line MeWo demonstrate two populations of binding sites of high affinity with dissociation equilibrium constants of Kd 4 X 10(-11) and 5 X 10(-10) M, respectively. Other cell lines of neural crest origin also show high-affinity binding heterogeneity. Exposure of the cell lines to 1 mM theophylline reversibly reduces the number of available NGF binding sites without influencing the affinities of binding. On the MeWo cell line, this is not related to the stages of the cell cycle or to production and release of a NGF-like molecule by the theophylline-exposed cells. These observations complement earlier studies of theophylline-induced alterations in NK cell sensitivity on the MeWo cell line, providing further evidence for cell surface phenotypic changes induced by a compound that promotes differentiation in melanocytes and melanoma cells. Future studies of cell surface phenomena involved in theophylline-induced NGF binding site disappearance may lead to a better understanding of the NGF receptor and its disappearance from certain embryonic cells of neural crest origin during differentiation.

Distinct effects of CD30 and Fas signaling in cutaneous anaplastic lymphomas: a possible mechanism for disease progression.

Lymphomatoid papulosis is part of a spectrum of CD30+ cutaneous lymphoproliferative disorders characterized by spontaneous tumor regression. The mechanism(s) of regression is unknown. In a recent study, a selective increase in CD30 ligand expression in regressing lesions of lymphomatoid papulosis and cutaneous CD30+ anaplastic large cell lymphoma was shown, suggesting that activation of the CD30 signaling pathway may be responsible for tumor regression, whereas no difference in Fas/Fas ligand expression was found between regressing and nonregressing lesions. Therefore we tested the effects of CD30 and Fas activation on three CD30+ cutaneous lymphoma cell lines (Mac-1, Mac-2 A, JK) derived from nonregressing tumors of two patients who had progressed from lymphomatoid papulosis to systemic anaplastic large cell lymphoma. To evaluate the effects of CD30 signaling, the cell lines were incubated with a CD30 agonistic antibody, HeFi-1. Proliferative responses, mitogen-activated protein kinase, and nuclear factor kappa B activities were determined with and without CD30 activation. Mac-1 and Mac-2 A showed increased proliferative responses to incubation with CD30 activating antibody, HeFi-1. Inhibition of the mitogen-activated protein kinase activity caused growth inhibition of the Mac-1, Mac-2 A, and JK cell lines. Activation of the Fas pathway induced apoptosis in all three cell lines. Taken together, these findings suggest that resistance to CD30-mediated growth inhibition provides a possible mechanism for escape of cutaneous anaplastic large cell lymphoma from tumor regression. Mitogen-activated protein kinase inhibitors are potential therapeutic agents for the treatment of advanced cutaneous anaplastic large cell lymphoma. J Invest Dermatol 115:1034-1040, 2000

Identification and characterization of a transcript for a novel Rac GTPase-activating protein in terminally differentiating 3T3-L1 adipocytes.

Using differential display, we sought to identify novel genes expressed in the early stages of 3T3-L1 adipocyte differentiation. A gene which we have named "band25" was identified, and a full-length cDNA sequence was assembled. Sequence analysis revealed that the 2842-bp cDNA encodes a putative 628-amino acid protein product, which is a member of the GTPase-activating protein (GAP) family. This gene may be the murine homolog of the human MgcRacGAP protein, which was identified in male germ cells. Other closely related proteins include the Drosophila protein Rotund, several chimerins, and the human breakpoint cluster region (Bcr) protein. These GAP proteins all specifically inactivate Rac, a member of the Ras-like family of proteins. A consensus sequence for a diacyl glycerol/phorbol ester-binding domain was also found in the Band25 sequence. The expression of band25 mRNA is regulated during the differentiation of both adipocytes and myoblasts. Its mRNA was shown to be expressed at a low level in confluent 3T3-L1 preadipocytes and in differentiated 3T3-L1 adipocytes. Expression of band25 was increased 15.5 fold by 24 h after the induction of differentiation, when 3T3-L1 cells undergo several rounds of postconfluent cell division. Expression was also high in growing 3T3-L1 and C2C12 cells but decreased progressively as C2C12 cells underwent differentiation. These observations suggest that the expression of band25 is growth regulated and that the protein could play a role in the regulation of growth-related processes.

The cell biology of ras-induced transformation: insights from studies utilizing an inducible hybrid oncogene system.

The c-Ha-ras oncogene has been implicated as a causative agent in the development of tumors in humans as well as mice. The molecular nature of the ras-induced tumorigenic process remains unclear, however. To address this question directly we have constructed a cell line which carries a zinc-inducible metallothionein-ras hybrid oncogene, transformant 212. Upon exposure to zinc for 24-48 hr, 212 cells assume a highly transformed morphology, concomitant with the induction of ras-expression. Natural killer cells constitute a subpopulation of lymphoid effector cells which have for a long time been hypothesized to be involved in the earliest stages of antitumor surveillance. Central to this hypothesis is the prediction that NK sensitivity arises during cellular transformation. By carrying out cytotoxicity assays against the 212 transformant, we showed that, indeed, increased sensitivity to NK-mediated lysis correlated with expression of the ras oncogene, which is consistent with the above hypothesis. We then addressed the question of the biochemical mechanism of ras-induced transformation. Owing to their similarity to G proteins, regulatory elements interposed between cell-surface receptors and their effector enzymes, it has been postulated that p21, the ras oncogene protein, mediates its transforming effects by constitutive activation of proliferative signal transduction pathways. We studied the effect of ras expression on the regulation of adenylate cyclase (A.C.), key enzyme of one such major pathway. We found that ras expression correlated with a dampening of responsiveness of A.C. to several stimuli, including hormones such as isoproterenol and other agents such as GMP-PNP, forskolin and fluoride-ion. Accumulation of cAMP as measured by RIA in intact cells, as basal or in response to stimulation of A.C. activity with forskolin, was also decreased (approximately 10-fold) with ras expression. Because the regulation of calcium, another important second messenger is dependent, in part, upon cAMP and GTP-binding proteins, we investigated the possible influence of ras expression on the intracellular concentration of calcium. Steady-state intracellular free Ca2+ concentration, as measured by fluorimetry, was indeed increased by approximately 50-125% in association with ras expression. Finally, we studied the possible influence of p21ras on protein kinase C (PKC), which is a key enzyme in the important signal transduction pathway of phosphatidylinositol lipid turnover. We assessed PKC activity directly, in a cell-free system, by measuring the ability of the enzyme to transfer radiolabelled phosphate from gamma-32P-ATP to histone, and exogenous substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Do NK cells play a role in anti-tumor surveillance?

A class of lymphocytes found in several mammalian species including man will kill cells of many tumor lines invitro(1-4). There is growing evidence, derived mainly from the work of Kiessling and co-workers, that these natural killer (NK) cells play a role in surveillance against tumor developmentin vivo. In this article John Roder and Tina Haliotis discuss a hypothesis for anti-tumor surveillance which integrates all of the potentially important immunological systems in the host and gives to NK cells the role of foremost barrier against developing tumors.

Regulation of natural antibody binding and susceptibility to natural killer cells through Zn(++)-inducible ras oncogene expression.

Changes in the natural resistance phenotype were examined for the 2H1, 10T 1/2 cells expressing the activated human H-ras oncogene under the transcriptional regulation of the zinc-inducible mouse metallothionein-I promoter. Culture of the cells in 50 microM ZnSO4 induced an increase in ras protein p21 levels which were maximal within 1 day. Natural-antibody (NAb) binding was significantly increased following 2 days of cell culture in ZnSO4 and continued to increase up to 4 days. The increased NAb binding returned to uninduced levels within 2 days following the removal of added zinc ions from the culture medium. The cells also exhibited a significant increase in natural killer (NK) cell sensitivity following 2 days in ZnSO4. This was maintained as long as the zinc was in the medium, but returned to uninduced levels within 1 day following its removal. The results show that NAb binding and susceptibility to NK cells increased following ras oncogene expression in 10T 1/2 cells and that both parameters were regulated by p21 expression. Repeated i.v. administration of whole-serum NAb prior to tumor inoculation reduced the number of early tumors following s.c. injection of Zn(++)-induced 2Hl cells into Zn(++)-treated C3H/HeN mice, consistent with an in vivo role for NAb in the defense against ras-transformed cells. In contrast, small but statistically significant reductions in NAb binding were observed following v-H-ras transformation of NIH 3T3 cells or v-src transformation of 10T 1/2. The data argue for an NAb- and NK-cell-susceptible phase of ras-induced tumor development which is a prerequisite for these mediators to contribute to a first line of defense against incipient neoplasia, and suggest that characteristics of the recipient cell and the transforming oncogene are important in determining the natural resistance phenotype.

Selective natural killer resistance in a clone of YAC lymphoma cells.

YAC lymphoma cells were treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and then cloned and subcloned. Of 51 clones, 3 were selected for further study. Ten-fold more natural killer (NK) effector cells were required to lyse YAC clone 6 and subclone 6-28 cells compared with clone 19 cells or the YAC parent cell line. The maximum plateau level of cytolysis of the NK-resistant (NKR) variants (20%) never approached that of the NK-sensitive (NKS) variants or YAC parental cells (60%) even after prolonged incubation (20 hr). NKR variants appeared with equal frequency (0.10) on cloning YAC cells that had not been treated with mutagen but these variants were highly unstable with respect to NK sensitivity and were not studied further. Cytolysis of both NKR and NKS lines was mediated by nylon-nonadherent asialo-GM1+ effector cells, and effectors from poly(I) . poly(C)-boosted mice preferentially lysed the NKS lines. The NKR alteration did not appear to change the NK target structure (NK-TS): (i) unlabeled NKR cells competed equally with NKS cells in reciprocal unlabeled-target competition assays; (ii) the frequency of target--effector conjugates was identical with NKR or NKS lines; and (iii) normal rabbit serum, which contains antibodies thought to react with the NK-TS, reacted equally against both NKR and NKS targets. The NKR alteration was selective for NK cells and did not result in a resistance to lysis in general; NKR and NKS variants were equally susceptible to (i) cytolysis mediated by alloimmune or lectin-dependent effector T cells and (ii) antibody- and complement-mediated lysis. These results are compatible with the hypothesis that the NKR variants have an altered acceptor site on the target cell membrane that normally binds the "lytic moiety" delivered by the effector cell.

Spontaneous and induced primary oncogenesis in natural killer (NK)-cell-deficient beige mutant mice.

Spontaneous tumor development and primary oncogenesis were compared in a large number of NK4-deficient, homozygous C57Bl/6-bg/bg mice and their NK normal, heterozygous +/bg littermate controls. In a group of 167 retired breeders followed for spontaneous tumors, the probability of survival for mice eventually dying with a tumor was greater for the NK-competent, +/bg than for the NK-deficient, homozygous C57BL/6-bg/bg mice (p = 0.0019), although the higher overall incidence of tumors in the bg/bg group (48%) was not significantly different from that in the +/bg group (37%). In the bg/bg group the incidence of tumor death appeared to increase relatively sharply in the 25- to 29-month age bracket compared to the fairly regular increase in incidence observed in the +/bg group. All the spontaneous tumors except 2 (discovered in +/bg mice) were classified histologically as widely disseminated malignant lymphomas. The other two were one squamous-cell carcinoma and one sarcoma. A total of 73 bg/bg mice injected s.c. with benzo[alpha]pyrene (BP) had a higher overall incidence of tumors (81%) (rhabdomyosarcomas) than 138 +/bg mice (64%) and in the largest group (0.3 mg, n = 85) the bg/bg group developed tumors, at a higher incidence (p = 0.01) and with a shorter latency (p = 0.025) than the +/bg group. On the other hand, mice injected with dimethylbenzanthracene or given 4 weekly doses of 160 rads of gamma irradiation showed no difference in overall tumor incidence. In addition, mice injected with various doses of DMBA-induced murine leukemia virus (DMBA-LV) also showed no significant difference in tumor incidence. Others have reported that some of these treatments (DMBA, split-dose irradiation) cause profound NK suppression, thereby reducing NK differences between the two groups of mice. These results suggest that a partial NK impairment in beige mutant mice early in life may lead to significantly greater rates of death with spontaneous malignant tumors late in life. Some primary oncogenesis treatments (BP) but not others (DMBA, split-dose irradiation, leukemia viruses) cause tumors with a greater incidence and shorter latency in beige mice. The results suggest, but do not prove, that NK cells play a role in surveillance against spontaneously arising, and possibly some types of carcinogen-induced, tumors.

Cellular ras gene activity is required for full neoplastic transformation by polyomavirus.

To investigate the role of ras gene activity in cellular transformation by polyomavirus, murine C3H10T1/2 cells were rendered ras deficient by transfection with an antisense ras gene construct. Ras deficiency resulted in a partial suppression of the polyomavirus-induced transformed phenotype. The production of viral middle T antigen and its association with pp60c-src, increased membrane-associated protein kinase C activity, and morphological transformation were unaffected by the downregulation of c-ras gene expression. On the other hand, stimulated proliferation, focus formation on confluent monolayers of normal cells, and colony formation in soft agar were all greatly reduced in cells containing reduced p21ras levels. It is concluded that ras gene activity is needed for full cell transformation by polyomavirus.

Ras modulates commitment and maturation of 10T1/2 fibroblasts to adipocytes.

The positive association of the ras oncogene with human cancer and the recognition that malignancy may, in part, represent the imbalance between cell proliferation and differentiation have generated intense interest in the potential role of ras in cell differentiation. We investigated this possibility utilizing as a model system the differentiation of the mesenchymal cell line C3H 10T1/2 (10T1/2) to adipocytes, and a series of transfectants of 10T1/2 cells in which the level of the ras gene product (p21ras; Ras) can be effectively up- or down-modulated. In agreement with previous reports, we found that 10T1/2 cultures, propagated in the resting state for several weeks, spontaneously convert to fat cells at a very low frequency. Downmodulation of endogenous p21ras levels, as a consequence of expression of antisense ras, markedly increased the rapidity and frequency of adipose conversion (6- to 10-fold), which was equivalent in magnitude to that effected by the potent differentiating agent 5-azacytidine. Conversely, overexpression of ras completely inhibited cell differentiation. In addition, adipocytes derived from antisense-ras expressing lines were characterized by a decrease in hormone responsiveness, as well as an apparent deficiency in attaining the terminally differentiated state. These findings suggest that Ras may be a negative regulator of the decision-making step of fibroblast differentiation to adipocytes. In addition, Ras may play an essential positive role in the transduction of hormonal signals necessary for full adipocyte maturation during later progression along the differentiation pathway.

Cellular ras gene activity is required for full neoplastic transformation by the large tumor antigen of SV40.

To investigate the role of the cellular ras gene product in neoplastic transformation by the SV40 large tumor antigen (SVLT), murine C3H10T1/2 cells were rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs or through the introduction of the dominant-negative mutant, ras(asn17). Consistent with previous results, SVLT-induced morphological transformation was unaffected by the down-regulation of c-ras gene product activity. On the other hand, colony formation in soft agar and tumorigenicity in nude mice were drastically reduced in c-Ras-deficient cells. In addition, SVLT expression in C3H10T1/2 cells led to increased c-Ras activity, as determined by an increase in the Ras-bound GTP/GTP + GDP ratio. These results suggest that c-Ras is required for full neoplastic transformation by SVLT.

v-Ras and v-Raf block differentiation of transformable C3H10T1/2-derived preadipocytes at lower levels than required for neoplastic transformation.

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiative insulin signaling pathway, we introduced a leu61-activated ras gene into a Ras-transformable, C3H10T1/2-derived preadipocytic cell line. The results demonstrate that rasleu61 expression in this line blocks differentiation and that this block appears at lower levels than required for full neoplastic transformation. In addition, to examine whether the inability of Rasleu61 to induce differentiation by replacing the insulin signal could be attributed to its transforming effect in this system, we examined the effect of Rasleu61 at levels below the baseline, by expressing rasleu61 in a series of preadipocytes which were rendered deficient in endogenous c-Ras activity. The results show that even very low Rasleu61 levels, insufficient to restore the growth rate of these cells to normal, blocked rather than enhanced differentiation, indicating that rasleu61 expression alone is not sufficient to promote adipocytic differentiation in this system, even in the absence of neoplastic transformation. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrored the v-Ras effects upon adipocytic differentiation and transformation.

Ras(leu61) blocks differentiation of transformable 3T3 L1 and C3H10T1/2-derived preadipocytes in a dose- and time-dependent manner.

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiation-promoting insulin signaling pathway, we introduced a leu61-activated ras gene into the Ras-transformable 3T3 L1 (ATCC CCL92.1) and a number of C3H10T1/2-derived preadipocytic cell lines. The results demonstrate a quantitative reciprocal regulation of differentiation and several transformation-associated properties in response to graded levels of ras gene expression, with the loss of differentiative capacity, morphological transformation, stimulation of proliferation, and anchorage-independent growth requiring increasing levels of Ras(leu61) protein. Furthermore, using novel, tightly regulatable 3T3 L1 transfectants, we demonstrated that Ras(leu61) effectiveness in blocking adipocytic differentiation is strictly dependent on the timing of its expression relative to cell growth arrest, with ras(leu61) expression being ineffective at inhibiting differentiation or inducing morphological transformation once the differentiative process has commenced. Moreover, rasleu61 induction failed to substitute for or enhance the c-Ras-dependent differentiative insulin signal, even under conditions in which it did not induce transformation. Therefore, although necessary for insulin signal transduction, the Ras signal alone is not sufficient to induce adipocytic differentiation in this system. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrored the Rasleu61 effects on adipocytic differentiation and transformation.

Expression of ras oncogene leads to down-regulation of protein kinase C.

The effect of mutated c-Ha-ras expression on Ca2+ and phospholipid-dependent protein kinase C (PKC) activity during the process of transformation was analysed using an inducible metallothionein-ras hybrid oncogene system. A close correlation was found between the timing of ras expression and the loss of PKC enzymatic activity measured in a cell-free system. Examination of the subcellular distribution of the enzyme in inducible and constitutive ras-transformants revealed that expression of ras was associated with an apparent translocation of PKC to the plasma membrane concomitant with down-regulation of PKC enzymatic activity in particulate as well as cytosolic fractions. Quantitation of PKC protein utilizing a PKC-specific antiserum showed that ras expression was associated with a decrease in the total amount of PKC protein present in the cell. We conclude that transformation by c-Ha-ras is accompanied by down-regulation of PKC activity and that the basis of this effect may, to a large extent, lie in the down-regulation of the amount of PKC protein.

A new immunodeficiency disorder in humans involving NK cells.

Immunodeficiency disorders have provided much information on the development and interaction of the various B and T lymphoid components in the immune system of man. As the lymphoid system becomes increasingly divided into functional subsets of cells it will be important to find immunodeficiencies affecting newly discovered cell types. Natural killer (NK) cells are a recently described but ill-defined subpopulation of lymphocytes which is thought to play an important part in surveillance against tumour development. Mice homozygous for the beige gene were found to have a selective deficiency in NK function and were more susceptible to transplantation of syngeneic tumours as predicted. We report here that patients carrying the analogous, autosomal recessive Chediak-Higashi (CH) gene have a profound defect in their ability to spontaneously lyse various tumour cells in vitro by either antibody-dependent or independent mechanisms. Since other cell-mediated cytolytic functions were relatively normal, these results suggest that the beige or Chediak-Higashi gene in both man and mouse controls NK function.