Blocking anaplerotic entry of glutamine into the TCA cycle sensitizes K-Ras mutant cancer cells to cytotoxic drugs.

Cancer cells undergo a metabolic transformation that allows for increased anabolic demands, wherein glycolytic and tricarboxylic acid (TCA) cycle intermediates are shunted away for the synthesis of biological molecules required for cell growth and division. One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis. With the loss of mitochondrial citrate, cancer cells rely on the 'conditionally essential' amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates. Although Q deprivation causes G1 cell cycle arrest in non-transformed cells, its impact on the cancer cell cycle is not well characterized. We report here a correlation between bypass of the Q-dependent G1 checkpoint and cancer cells harboring K-Ras mutations. Instead of arresting in G1 in response to Q-deprivation, K-Ras-driven cancer cells arrest in either S- or G2/M-phase. Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation. Blocking anaplerotic utilization of Q mimicked Q deprivation--causing S- and G2/M-phase arrest in K-Ras mutant cancer cells. Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel. These data suggest that disabling of the G1 Q checkpoint could represent a novel vulnerability of cancer cells harboring K-Ras and possibly other mutations that disable the Q-dependent checkpoint.

HIF alpha expression in VHL-deficient renal cancer cells is dependent on phospholipase D.

Loss of the von Hippel-Lindau (VHL) tumor suppressor gene contributes to proliferative disorders including renal cell carcinoma. The consequence of VHL loss is increased levels of hypoxia-inducible factor-alpha (HIFalpha), which is targeted for proteolytic degradation by the VHL gene product pVHL. HIF is a transcription factor that increases the expression of factors critical for tumorigenesis in renal cell carcinoma. We report here another regulatory component of HIFalpha expression in renal cancer cells. Phospholipase D (PLD), which is commonly elevated in renal and other cancers, is required for elevated levels of both HIF1alpha and HIF2alpha in VHL-deficient renal cancer cells. The induction of both HIF1alpha and HIF2alpha by hypoxic mimetic conditions was also dependent on PLD in renal cancer cells with restored pVHL expression. The effect of PLD activity upon HIFalpha expression was at the level of translation. PLD activity also provides a survival signal that suppresses apoptosis induced by serum deprivation in the renal cancer cells. Suppression of HIF2alpha has been shown to reverse tumorigenesis with renal cancer cells. The finding here that HIF2alpha expression is dependent on PLD in renal cancer cells suggests that targeting PLD signals may represent an alternative therapeutic strategy for targeting HIF2alpha in renal cancers where HIF2alpha is critical for tumorigenesis and elevated PLD activity is common.

Defective TGF-beta signaling sensitizes human cancer cells to rapamycin.

mTOR, the mammalian target of rapamycin, is a critical target of survival signals in many human cancers. In the absence of serum, rapamycin induces apoptosis in MDA-MB-231 human breast cancer cells. However, in the presence of serum, rapamycin induces G(1) cell cycle arrest-indicating that a factor(s) in serum suppresses rapamycin-induced apoptosis. We report here that transforming growth factor-beta (TGF-beta) suppresses rapamycin-induced apoptosis in serum-deprived MDA-MB-231 cells in a protein kinase Cdelta (PKCdelta)-dependent manner. Importantly, if TGF-beta signaling or PKCdelta was suppressed, rapamycin induced apoptosis rather than G(1) arrest in the presence of serum. And, if cells were allowed to progress into S phase, rapamycin induced apoptosis in the presence of serum. BT-549 and MDA-MB-468 breast, and SW-480 colon cancer cells have defects in TGF-beta signaling and rapamycin induced apoptosis in these cells in the presence of either serum or TGF-beta. Thus, in the absence of TGF-beta signaling, rapamycin becomes cytotoxic rather than cytostatic. Importantly, this study provides evidence indicating that tumors with defective TGF-beta signaling--common in colon and pancreatic cancers--will be selectively sensitive to rapamycin or other strategies that target mTOR.

Mutant p53 in MDA-MB-231 breast cancer cells is stabilized by elevated phospholipase D activity and contributes to survival signals generated by phospholipase D.

p53 is the most commonly mutated gene in human cancer. Although the loss of tumor suppressor functions for p53 in tumorigenesis is well characterized, gain-of-function p53 mutations observed in most cancers are not as widely appreciated. The human breast cancer cell line MDA-MB-231, which has high levels of a mutant p53, has high levels of phospholipase D (PLD) activity, which provides a survival signal in these cells when deprived of serum growth factors. We report here that the mutant p53 in MDA-MB-231 cells is stabilized by the elevated PLD activity in these cells. Surprisingly, the survival of MDA-MB-231 cells deprived of serum was dependent on the mutant p53. These data indicate that a mutant p53, stabilized by elevated PLD activity, can contribute to the suppression of apoptosis in a human breast cancer cell line and suggest a rationale for the selection of p53 mutations early in tumorigenesis to suppress apoptosis in an emerging tumor.

Transformation of cells overexpressing a tyrosine kinase by phospholipase D1 and D2.

Phospholipase D (PLD) activity is elevated in response to most mitogenic signals. Two mammalian PLD genes (PLD1 and PLD2) have been cloned and their gene products have been characterized. PLD1 is a downstream target of the Ras/RalA GTPase cascade implicated in mitogenic and oncogenic signaling. Consistent with a role in mitogenic signaling, elevated expression of PLD1 transforms cells overexpressing the epidermal growth factor (EGF) receptor (EGFR). However, PLD2 colocalizes with the EGFR in caveolin-enriched light membrane microdomains. We therefore investigated whether PLD2 could also contribute to the transformation of cells overexpressing a tyrosine kinase. We report here that elevated expression of PLD2 transforms rat fibroblasts overexpressing either the EGFR or c-Src. Since overexpression of a tyrosine kinase is a common genetic alteration in several human cancers, these data suggest that elevation of either PLD1 or PLD2 may contribute to the progression to a malignant phenotype in cells with elevated tyrosine kinase activity.

The impact of psychiatric diagnosis and family system relationship on problems identified by families coping with a mentally ill member.

The purpose of this study was to explore how psychiatric diagnosis and family relationships relate to problems identified by participants in three one-day public family psychoeducation workshops for families with a member with a serious mental illness. Workshop participants generated lists of problems they had faced, which were coded into eleven categories. Logistic regression models predicting listing of categories were developed based on ill member (diagnosis, sex, treatment compliance) and family member (sex, age, relationship to the ill member) characteristics. For models predicting content category from ill member characteristics, only denial/noncompliance and interpersonal/social categories were significantly predictive as dependent variables. For models predicting content categories from family member characteristics, only the resources/benefits model was predictive. The significant findings, in conjunction with the important negative results, suggest implications for further development of family intervention models. Building on previous research, groups composed of families coping with more than a single diagnosis and including a variety of family member relationships have the potential to reach consensus on curriculum topics.

RalA mediates v-Src, v-Ras, and v-Raf regulation of CD44 and fibronectin expression in NIH3T3 fibroblasts.

Oncogenic transformation of fibroblasts by v-Src and v-Ras is often associated with downregulation of fibronectin (FN) and increased expression of CD44, a receptor for hyaluronan. Both v-Src and v-Ras as well as v-Raf activate phospholipase D through the small GTPase, RalA, an important mediator of transformation and tumorigenesis in vivo. We have therefore investigated whether RalA is involved in the downregulation of FN and overproduction of CD44 upon oncogenic transformation. We report here that compared to untransfected cells NIH3T3 cells transformed by v-Src, v-Ras, or v-Raf have reduced levels of FN and increased levels of CD44. Moreover, the ability to form extracellular FN fibrils was significantly reduced in the oncogene-transformed cells compared to parental controls. Coexpression of the dominant negative S28N-RalA mutant restored the levels of CD44 and FN and the capacity of v-Src-, v-Ras-, and v-Raf-expressing cells to form extracellular FN fibrils, to those observed in NIH3T3 cells. The data presented here show a novel regulatory role for RalA, which is required for tumor formation in transformed NIH3T3 cells, in mediating the signal transduction pathway activated by v-Src, v-Ras, and v-Raf, that leads to FN downregulation and CD44 overexpression.

Novel tumor-promoting property of tamoxifen.

The tumor-promoting phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) cooperates with c-Src overexpression to transform rat fibroblasts. TPA transforms c-Src-overexpressing cells by depleting the delta isoform of protein kinase C (PKCdelta). Tamoxifen, which has both estrogen-mimetic and estrogen-antagonist properties, has been widely used to improve the prognosis of breast cancer patients. However, with extended use, there is an increased risk for endometrial and other cancers that can be observed within 10 years of treatment. We report here that tamoxifen, similar to TPA, cooperates with c-Src overexpression to transform 3Y1 rat fibroblasts. Tamoxifen induced both DNA synthesis and anchorage-independent cell proliferation in c-Src-overexpressing, but not in parental, 3Y1 rat fibroblasts. Tamoxifen also induced an association between c-Src and PKCdelta that resulted in the tyrosine phosphorylation and down-regulation of PKCdelta. These phenotypes were not induced by estrogen, indicating that the effect of tamoxifen was in addition to any estrogen-mimetic effects. Thus, in addition to the hyperplasia-inducing capability of an estrogen-mimetic, tamoxifen has an additional tumor-promoting capability similar to that of TPA. The dual tumor-promoting capability of both estrogen- and TPA-mimetic properties for tamoxifen may contribute to the increased incidence of endometrial cancers observed in the relatively short exposure period of <10 years.

Role for phospholipase D in receptor-mediated endocytosis.

In response to epidermal growth factor (EGF), the EGF receptor is endocytosed and degraded. A substantial lag period exists between endocytosis and degradation, suggesting that endocytosis is more than a simple negative feedback. Phospholipase D (PLD), which has been implicated in vesicle formation in the Golgi, is activated in response to EGF and other growth factors. We report here that EGF receptor endocytosis is dependent upon PLD and the PLD1 regulators, protein kinase C alpha and RalA. EGF-induced receptor degradation is accelerated by overexpression of either wild-type PLD1 or PLD2 and retarded by overexpression of catalytically inactive mutants of either PLD1 or PLD2. EGF-induced activation of mitogen-activated protein kinase, which is dependent upon receptor endocytosis, is also dependent upon PLD. These data suggest a role for PLD in signaling that facilitates receptor endocytosis.

Service use over time and achievement of stable housing in a mentally ill homeless population.

OBJECTIVE: Service use among clients of a multiservice agency serving homeless persons with severe mental illness was examined to determine whether patterns of service use reflected two stages in an adaptation of the transtheoretical model of change. In the adapted model, change occurs in five stages-pre-engagement, contemplation, engagement, the strategic moment, and consolidation. It was hypothesized that rates of service use would be highest immediately after clients obtained housing (the strategic moment) and would decrease in the months afterward (consolidation stage), with the greatest decreases occurring immediately after housing was obtained. METHODS: Service use data were collected for two groups: a housed group of 58 clients who had obtained and sustained stable housing for at least 24 consecutive months at the time of sampling and an unhoused group of 55 clients who were matched with the housed clients on month of service entry. Total service use and use of three service types-a drop-in center, counseling, and health services-were examined to test the hypotheses. It was hypothesized that use of services by the unhoused group would show a consistent linear decline rather than a two-stage decline. Linear spline regression using bootstrap sampling methods was used to fit service use data for both groups. RESULTS: The two-stage solution significantly modeled the patterns of service use by the housed but not the unhoused clients, supporting the hypotheses. For the housed group, use of the drop-in center and counseling fit the model, and use of health services did not. CONCLUSIONS: The results provide limited support for the hypothesized five-stage model for achieving change.

Epidemiology of Reye's syndrome, United States, 1991-1994: comparison of CDC surveillance and hospital admission data.

This investigation describes the epidemiology of Reye's syndrome (RS) during 1991-1994 and compares two different sources of information in the United States. Estimates of the incidence of RS from the Centers for Disease Control and Prevention (CDC) are compared with hospital inpatient data from approximately one third of the hospitals from HCIA, Inc. During 1991-1994, 48 RS cases were reported to the CDC and 93 RS hospitalizations based on HCIA data. When the HCIA data are extrapolated to the US population, there were an estimated 284 hospitalizations. Cases reported from both data sources were similar in distribution by onset, age, and sex. CDC data probably underestimate the incidence of RS due to incomplete reporting and HCIA data may overestimate it because not all cases were known to meet the CDC case definition. The true annual incidence of RS during the study years was probably between 0.2 and 1.1 cases per million population <18 years of age.

Mitogenic phospholipase D activity is restricted to caveolin-enriched membrane microdomains.

Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of several signaling oncogenes. PLD activity is also elevated in response to peptide growth factors, indicating that PLD likely plays an important role in mitogenic signaling. Many proteins that mediate mitogenic signaling are localized in caveolin-enriched membrane microdomains (CEMMs). We report here that the elevated PLD activity in NIH 3T3 cells transformed by activated oncogenic forms of Src, Ras, and Raf is largely restricted to the CEMMs. Likewise, the PLD activity stimulated by epidermal growth factor is also restricted to the CEMMs. Although both PLD1 and PLD2 were found in CEMMs, neither was particularly enriched in the CEMMs of the transformed relative to the parental cells, indicating that it is the specific activity of PLD that is increased in the CEMMs. An apparent PLD substrate specificity in transformed cells for phosphatidylcholine lacking arachidonate acyl groups is also explained by the localization of activity in the CEMMs where [(3)H]arachidonate-labeled PC was excluded. These data indicate that mitogenic signals through PLD are initiated in CEMMs where many signaling molecules colocalize.

A low molecular weight factor from dividing cells activates phospholipase D in caveolin-enriched membrane microdomains.

Phospholipase D (PLD) activity is elevated in Ras-transformed NIH 3T3 cells. This difference in PLD activity between Ras-transformed and nontransformed parental cells disappeared in isolated membranes from these cells. In reconstitution experiments, heat-denatured cytosolic fractions from Ras-transformed, but not parental, NIH 3T3 cells elevated PLD activity in isolated membranes. This heat-resistant PLD-stimulating activity from the Ras-transformed cells was sensitive to proteases and passed through a 1-kDa MW cutoff membrane, suggesting that the factor is a peptide of less than 10 amino acids. The ability of this PLD-stimulating factor, designated PLD-SF, to elevate PLD activity in isolated membranes was restricted to the caveolin-enriched light membranes, where many signaling molecules are localized. PLD-SF was also elevated in v-Src- and v-Raf-transformed cells and in serum-stimulated NIH 3T3 cells. PLD-SF was detected in a variety of rat tissues but was highest in testes, where a large percentage of cells are dividing. A similar low molecular weight PLD-stimulating activity was found in actively dividing, but not stationary yeast, cells. The data here provide evidence for a highly conserved PLD-stimulating peptide that is elevated in response to mitogenic stimuli.

An EGF receptor/Ral-GTPase signaling cascade regulates c-Src activity and substrate specificity.

c-Src is a membrane-associated tyrosine kinase that can be activated by many types of extracellular signals, and can regulate the function of a variety of cellular protein substrates. We demonstrate that epidermal growth factor (EGF) and beta-adrenergic receptors activate c-Src by different mechanisms leading to the phosphorylation of distinct sets of c-Src substrates. In particular, we found that EGF receptors, but not beta(2)-adrenergic receptors, activated c-Src by a Ral-GTPase-dependent mechanism. Also, c-Src activated by EGF treatment or expression of constitutively activated Ral-GTPase led to tyrosine phosphorylation of Stat3 and cortactin, but not Shc or subsequent Erk activation. In contrast, c-Src activated by isoproterenol led to tyrosine phosphorylation of Shc and subsequent Erk activation, but not tyrosine phosphorylation of cortactin or Stat3. These results identify a role for Ral-GTPases in the activation of c-Src by EGF receptors and the coupling of EGF to transcription through Stat3 and the actin cytoskeleton through cortactin. They also show that c-Src kinase activity can be used differently by individual extracellular stimuli, possibly contributing to their ability to generate unique cellular responses.

Phospholipase D and RalA cooperate with the epidermal growth factor receptor to transform 3Y1 rat fibroblasts.

3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) receptor (EGFR cells) become transformed when treated with EGF. A common response to oncogenic and mitogenic stimuli is elevated phospholipase D (PLD) activity. RalA, a small GTPase that functions as a downstream effector molecule of Ras, exists in a complex with PLD1. In the EGFR cells, EGF induced a Ras-dependent activation of RalA. The activation of PLD by EGF in these cells was dependent upon both Ras and RalA. In contrast, EGF-induced activation of Erk1, Erk2, and Jun kinase was dependent on Ras but independent of RalA, indicating divergent pathways activated by EGF and mediated by Ras. The transformed phenotype induced by EGF in the EGFR cells was dependent upon both Ras and RalA. Importantly, overexpression of wild-type RalA or an activated RalA mutant increased PLD activity in the absence of EGF and transformed the EGFR cells. Although overexpression of PLD1 is generally toxic to cells, the EGFR cells not only tolerated PLD1 overexpression but also became transformed in the absence of EGF. These data demonstrate that either RalA or PLD1 can cooperate with EGF receptor to transform cells.

Antagonistic effects of protein kinase C alpha and delta on both transformation and phospholipase D activity mediated by the epidermal growth factor receptor.

Downregulation of protein kinase C delta (PKC delta) by treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) transforms cells that overexpress the non-receptor class tyrosine kinase c-Src (Z. Lu et al., Mol. Cell. Biol. 17:3418-3428, 1997). We extended these studies to cells overexpressing a receptor class tyrosine kinase, the epidermal growth factor (EGF) receptor (EGFR cells); like c-Src, the EGF receptor is overexpressed in several human tumors. In contrast with expectations, downregulation of PKC isoforms with TPA did not transform the EGFR cells; however, treatment with EGF did transform these cells. Since TPA downregulates all phorbol ester-responsive PKC isoforms, we examined the effects of PKC delta- and PKC alpha-specific inhibitors and the expression of dominant negative mutants for both PKC delta and alpha. Consistent with a tumor-suppressing function for PKC delta, the PKC delta-specific inhibitor rottlerin and a dominant negative PKC delta mutant transformed the EGFR cells in the absence of EGF. In contrast, the PKC alpha-specific inhibitor Go6976 and expression of a dominant negative PKC alpha mutant blocked the transformed phenotype induced by both EGF and PKC delta inhibition. Interestingly, both rottlerin and EGF induced substantial increases in phospholipase D (PLD) activity, which is commonly elevated in response to mitogenic stimuli. The elevation of PLD activity in response to inhibiting PKC delta, like transformation, was dependent upon PKC alpha and restricted to the EGFR cells. These data demonstrate that PKC isoforms alpha and delta have antagonistic effects on both transformation and PLD activity and further support a tumor suppressor role for PKC delta that may be mediated by suppression of tyrosine kinase-dependent increases in PLD activity.

RalA requirement for v-Src- and v-Ras-induced tumorigenicity and overproduction of urokinase-type plasminogen activator: involvement of metalloproteases.

Overproduction of urokinase-type plasminogen activator (uPA) and metalloproteases (MMPs) is strongly correlated with tumorigenicity and with invasive and metastatic phenotypes of human and experimental tumors. We demonstrated previously that overproduction of uPA in tumor cells is mediated by a phospholipase D (PLD)- and protein kinase C-dependent mechanism. The oncogenic stimulus of v-Src and v-Ras results in the activation of PLD, which is dependent upon the monomeric GTPase RalA. We have therefore investigated whether RalA plays a role in uPA and MMP overproduction that is observed in response to oncogenic signals. We report here that NIH3T3 cells transformed by both v-Src and v-Ras, constitutively overproduce uPA and that expression of a dominant negative RalA mutant (S28N) blocks overproduction of uPA in both the v-Src-and v-Ras-transformed cells. v-Src and v-Ras also induced an upregulation of the activity of MMP-2 and MMP-9 as detected by zymograms, however only the v-Src induction correlated with MMP protein levels detected by Western blot analysis. The dominant negative RalA mutant blocked increased MMP-2 and 9 overproduction induced by v-Src, but not the increased activity of MMP-2 and 9 induced by v-Ras. And, consistent with a role for the RalA/PLD pathway in mitogenesis and tumor development, the dominant negative RalA mutant completely blocked tumor formation by v-Src- and v-Ras-transformed NIH3T3 cells injected subcutaneously in syngeneic mice. The data presented here implicate RalA and PLD as signaling mediators for tumor formation and protease production by transformed cells.

ETS transcription factors regulate an enhancer activity in the third intron of TNF-alpha.

We describe an enhancer site in the third intron of tumor necrosis factor alpha (TNF-alpha). A reporter construct containing the 5'-flanking region of the mouse TNF-alpha gene displayed weak activity when transfected into RAW264.7 macrophage-like cells. The addition of the third intron of TNF-alpha to this construct resulted in an enhancement of CAT protein. This enhancement was eliminated if a conserved 20-bp sequence was removed from the intron or if a dominant-negative ets-binding factor was co-transfected with the reporter gene. Mutations of this site that destroyed potential ets transcription factor binding sites had reduced transcriptional activity. The major transcription factor that bound to the oligonucleotide was confirmed to be GABP by supershift and competition analysis. In RAW264.7 cells, the binding was constitutive, however, in bone marrow-derived macrophages binding activity was shown to be interferon-gamma inducible. This may imply a role for ets transcription factors in the production of TNF-alpha.