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To determine the tolerability of oral maintenance topotecan when administered to patients with advanced ovarian, fallopian tube, and primary peritoneal serous cancers who have achieved a complete clinical response after first-line platinum-based therapy. Oral topotecan was given at a starting dose of 0.4 mg/m(2)/dose, twice a day (BID) for 21 consecutive days out of 28 days. The dose was subsequently increased to 0.5 mg/m(2)/dose, twice a day as tolerated. If the patient experienced toxicities during cycle 1 or subsequent cycles, doses were delayed and/or reduced. The lowest dose allowed on protocol was 0.3 mg/m(2)/dose twice daily. Thirteen patients were enrolled in the study, representing a total of fifty-nine cycles of oral topotecan. The starting dose of 0.4 mg/m(2) by mouth (PO) BID for 21 days was generally difficult for patients to tolerate, usually due to progressive anemia and fatigue, and a dose reduction to 0.3 mg/m(2) was necessary in 10/13 patients. A median of six cycles was administered, although 6 of 13 patients could not tolerate the planned 6 cycles due to toxicity. Hematologic toxicity was the most common side effect, although there were no episodes of febrile neutropenia. Diarrhea was the most common nonhematologic side effect, occurring in 8 of 13 patients. Six patients were removed from the study prior to completing the planned six cycles of therapy, after receiving a median number of 2.5 cycles of treatment. This dose and schedule of oral topotecan does not appear to be feasible in this patient population.

The IGF axis has documented growth-promoting effects in various malignancies, but its role in epithelial ovarian cancer (EOC) has not been adequately examined. We studied the expression of the IGF axis genes in relation to outcome in EOC. Microarray expression profiles from 64 patients with advanced-stage EOC were used. Two multi-gene subsets were chosen, one upstream of the IGF receptor ('IGF family') and the other downstream of the IGF receptor ('IGF signaling pathway'), and analyzed in relation to survival. In addition, expression patterns of the two gene subsets were analyzed in relation to favorable and unfavorable prognosis categories identified in a previous study by whole-genome expression profiling. In a gene-by-gene analysis, IGF binding protein 4 and IGF-II receptor gene expression was inversely associated with survival. Using hierarchical clustering, the two multi-gene subsets separated the patient cohort into two groups with different median survival (IGF family: 33 vs 63 months, P=0.02 and IGF signaling pathway: 41 vs 63 months, P=0.05). Furthermore, the two multi-gene subsets were differentially expressed between the previously defined favorable and unfavorable prognosis tumors (Kolmogorov-Smirnov permutation: P=0.0005 and 0.003 for the IGF family and signaling pathway respectively), and individual genes (including IGF-I, IGF-I receptor, and several genes downstream of the receptor) were overexpressed in unfavorable prognosis tumors (permutation P<0.05). The expression patterns of several genes in the IGF axis are associated with survival in EOC, and expression changes of these genes may be underlying previously proposed microarray-derived clinical prognostic models. Future studies are needed to more precisely determine the diagnostic and potential therapeutic significance of these findings.

Purpose: We investigated whether tumor tissue obtained at diagnosis expresses a specific gene profile that is predictive of findings at second-look surgery in patients with epithelial ovarian cancer (EOC)., Patients and Methods: Tumor tissue obtained at the time of diagnosis was profiled with oligonucleotide microarrays. Class prediction analysis was performed in a training set of 24 patients who had undergone a second-look procedure. The resultant predictive signature was then tested on an independent validation set comprised of 36 patients., Results: A 93-gene signature referred to as the Chemotherapy Response Profile (CRP) was identified through its association with pathologic complete response. When applied to a separate validation set, the CRP distinguished between patients with unfavorable versus favorable overall survival (median 41 months v not yet reached, respectively, log-rank P = .007), with a median follow-up of 52 months. The signature maintained independent prognostic value in multivariate analysis, controlling for other known prognostic factors such as age, stage, grade, and debulking status. There was no genetic overlap between the CRP and our previously described Ovarian Cancer Prognostic Profile (OCPP), which demonstrated similar prognostic value. The combination of the CRP and OCPP yielded better prognostic discrimination then either profile alone. Genes present in the CRP include BAX, a proapoptotic protein previously associated with chemotherapy response in ovarian cancer., Conclusion: Identification of a gene expression profile based on pathologic response in EOC provides independent prognostic information and offers potential insights into the mechanism of drug resistance. Efforts to identify a more tailored profile using selected genes from both the CRP and OCPP are underway.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in adult women. The most easily identifiable riskfactor is a strong family history of either ovarian or breast cancer; that may indicate the presence of an inherited germ-line mutation in either BRCA-1 or BRCA-2. Common symptoms, such as abdominal bloating and early satiety, indicate more advanced disease, involving the upper abdomen and present in approximately 70% of patients at the time of diagnosis. Physical examination often reveals the presence of a pelvic mass, which is best evaluated by transvaginal ultrasound (TVU) for confirmation. Exploratory laparotomy is required for histologic confirmation, staging and tumor debulking and should be performed by a surgeon trained in these aspects of ovarian cancer management. Patients with early-stage disease, limited to the ovary or pelvis (stages I and II, respectively), have survival in the 80-95% range, whereas the survival of patients with disease involving the upper abdomen or beyond (stages III and IV, respectively) is 10-30%. Because of the propensity of EOC to spread beyond the confines of the ovary, the majority of patients will require postoperative chemotherapy in an attempt to eradicate residual disease. For selected patients with early-stage disease, confined to the ovary, such as those with well-differentiated, completely encapsulated tumors (e.g., stage IA, grade 1), no further treatment is necessary in view of excellent survival after surgery alone. For patients with higher-risk early-stage disease (e.g., those with pelvic extension, capsular rupture or involvement, positive washings, ascites or high-grade lesions) and for patients with advanced-stage disease (stages III and IV), postoperative combination chemotherapy with a taxane and platinum combination is the standard of care. Such treatment is capable of inducing responses in > 70% of patients with residual EOC and is also capable of prolonging both disease-free and overall survival. Unfortunately, despite an initial response to chemotherapy in the majority of patients, relapse is afrequent problem and is often detected by a rise in the serum tumor marker CA-125 in the absence of symptoms or signs of disease by physical examination or radiographic studies. In such cases, a hormonal maneuver is oftentimes considered in order to avoid the toxic effects of chemotherapy when the patient is asymptomatic and the goal of treatment is largely palliation, although eventually the development of clinical progression mandates the institution of second-line chemotherapy. If the treatment-free interval is > 6 months from the completion of first-line treatment, rechallenge with platinum-based chemotherapy is a reasonable first step. For those patients who develop resistance to second-line platinum or who have difficulty tolerating this agent, multiple other options are available for relapse management, including liposomal doxorubicin, topotecan, gemcitabine and etoposide per os. Eventually the disease becomes resistant to multiple chemotherapy agents, and reorienting management toward supportive care and pain control is necessary. Ongoing efforts to identify more effective multiagent first-line regimens, to develop more effective strategies for early detection and to incorporate agents with novel mechanisms of action, such as antiangiogenesis compounds, hold promise.

Objective: To determine the activity and tolerability of weekly docetaxel in patients with platinum-resistant mullerian origin tumors., Methods: Patients with persistent disease, or those recurring less than 6 months after receiving platinum-containing therapy, were eligible for this phase II study. Docetaxel was initially administered at a dose of 40 mg/m(2) on days 1, 8, and 15, with a cycle length of 28 days. This starting dose was subsequently reduced to 30 mg/m(2) due to toxicity. Dexamethasone prophylaxis was administered at a dose of 4 mg PO every 12 hours for 3 doses, starting 12 hours before each dose of docetaxel., Results: Thirty-two patients were enrolled, with a median age of 59 years. The majority of patients received a median of 3 prior regimens, with 45% of the study group having received 4 or more prior regimens. The overall response rate in 29 evaluable patients was 6.9%, with no complete responses. Seventeen percent of patients experienced stable disease. Dose reduction or delay was required in 10 of the first 22 patients enrolled, prompting a reduction in the starting dose to 30 mg/m(2). Hematologic toxicity was generally tolerable, and no patient experienced febrile neutropenia. Non-hematologic toxicity was generally grade 1 in nature, although a combination of multiple low grade toxicities occurring in an individual patient oftentimes mandated dose reduction., Conclusions: Weekly docetaxel demonstrated modest activity in a heavily pre-treated, platinum-resistant population. A starting docetaxel dose of 30 mg/m(2) would be reasonable for future studies exploring the utility of weekly dosing in less heavily pre-treated patients.

Purpose: Currently available clinical and molecular prognostic factors provide an imperfect assessment of prognosis for patients with epithelial ovarian cancer (EOC). In this study, we investigated whether tumor transcription profiling could be used as a prognostic tool in this disease., Methods: Tumor tissue from 68 patients was profiled with oligonucleotide microarrays. Samples were randomly split into training and validation sets. A three-step training procedure was used to discover a statistically significant Kaplan-Meier split in the training set. The resultant prognostic signature was then tested on an independent validation set for confirmation., Results: In the training set, a 115-gene signature referred to as the Ovarian Cancer Prognostic Profile (OCPP) was identified. When applied to the validation set, the OCPP distinguished between patients with unfavorable and favorable overall survival (median, 30 months v not yet reached, respectively; log-rank P = .004). The signature maintained independent prognostic value in multivariate analysis, controlling for other known prognostic factors such as age, stage, grade, and debulking status. The hazard ratio for death in the unfavorable OCPP group was 4.8 (P = .021 by Cox proportional hazards analysis)., Conclusion: The OCPP is an independent prognostic determinant of outcome in EOC. The use of gene profiling may ultimately permit identification of EOC patients appropriate for investigational treatment approaches, based on a low likelihood of achieving prolonged survival with standard first-line platinum-based therapy.

Objectives: The aim of this study was to assess the feasibility of combining docetaxel, carboplatin, and gemcitabine (DoCaGem) as first-line treatment of ovarian and other müllerian origin tumors., Methods: Four dose levels were planned for this phase I trial. Treatment consisted of carboplatin on day 1 only, docetaxel on days 1 and 8, and gemcitabine on days 1 and 8, every 21 days., Results: Nineteen patients were enrolled, of whom 18 were evaluable for toxicity. The first 5 patients enrolled at dose level I (carboplatin AUC 5, 30 mg/m(2) docetaxel, 800 mg/m(2) gemcitabine) experienced no dose-limiting toxicity. At dose level II, 4/4 evaluable patients experienced significant bone marrow suppression that required dose reduction and/or dose delay. Further dose escalation was not attempted, and 9 additional patients were enrolled in dose level I. Of the 67 cycles administered on dose level I, day 8 treatment could not be administered due to bone marrow suppression in 16 cycles (24%), and prolongation of the cycle length from day 22 to day 29 was required in 13 cycles (19%). There were no episodes of febrile neutropenia in evaluable patients and no treatment-related deaths. Grade 3 nonhematologic toxicity (fatigue) occurred in 1 cycle. Response was determined in the 14 evaluable patients who tolerated 4 or more cycles of therapy, with 11 obtaining a complete clinical response and 3 obtaining a partial clinical response., Conclusions: The DoCaGem regimen is highly active, but myelosuppression at dose level I prevents full dose delivery. Other strategies to combine these three active agents are reasonable to explore.

Adult granulosa cell tumor (GCT) of the ovary is oftentimes a hormonally active, stromal cell neoplasm that is distinguished by its ability to secrete sex steroids such as estrogen. Patients may present with vaginal bleeding caused by endometrial hyperplasia or uterine cancer as a result of prolonged exposure to tumor-derived estrogen. In addition, GCT is a vascular tumor that may occasionally rupture and result in abdominal pain, hemoperitoneum, and hypotension, mimicking an ectopic pregnancy in younger patients. GCT is usually associated with a mass on pelvic examination that is subsequently confirmed on ultrasonography. Surgery is required for definitive tissue diagnosis, staging, and tumor debulking. In older women, a total abdominal hysterectomy and bilateral salpingooophorectomy are typically performed. In women of childbearing age, a more conservative unilateral salpingo-oophorectomy may be performed, assuming that careful staging reveals that the disease has not extended outside of the involved ovary and that a concomitant uterine cancer has been excluded. Survival of patients with GCT is generally excellent because most patients present with early-stage disease, although certain high-risk patient groups may be identified. Stage is the most important prognostic factor, with a higher risk of relapse being associated with stages II through IV disease. In addition, patients with stage I disease associated with features such as large tumor size, high mitotic index, or tumor rupture may also be at higher risk in some series. The value of postoperative adjuvant therapy for high-risk patients has not been investigated by prospective randomized trials, which are difficult to perform because of the rarity of this tumor. Nonetheless, the use of adjuvant chemotherapy or radiation has sometimes been associated with prolonged disease-free survival in patients with high-risk features. Because of the propensity of GCT to recur years after initial diagnosis, prolonged surveillance with serial physical examination and serum tumor markers such as estradiol and inhibin is reasonable.

Peroxisome proliferator activated receptor gamma (PPARgamma) is a nuclear hormone receptor that has been shown to regulate differentiation and cell growth. Studies of the differentiative effects of PPARgamma agonists on several cancer cell lines led to the hypothesis that dysfunction of PPARgamma contributes to tumorigenesis. These functional observations were strengthened by genetic evidence: somatic loss-of-function mutations in PPARG, encoding PPARgamma, in sporadic colorectal carcinomas and somatic translocation of PAX8 and PPARG in follicular thyroid carcinoma. Recently overrepresentation of the H449H variant was found in a cohort of American patients with glioblastoma multiforme. The glioblastoma multiforme data suggest that PPARG contributes common, low-penetrance alleles for cancer susceptibility. To test this hypothesis in a broader range of cancers we examined a series of carcinomas of the cervix, endometrium, ovary, prostate, and kidney for germline sequence variation in PPARG. In addition to the two common sequence variants, P12A and H449H, there were five other sequence variants. P12A alleles were underrepresented in renal cell carcinoma patients compared to country-of-origin race-matched controls (3.75% vs. 12.1%, P<0.04). In contrast, the H449H variant was overrepresented in individuals with endometrial carcinoma compared to controls (14.4% vs. 6.25%, P<0.02). These observations lend genetic evidence consistent with our hypothesis that PPARG serves as a common, low-penetrance susceptibility gene for cancers of several types, especially those epidemiologically associated with obesity and fat intake.

PTEN (MMAC1/TEP1), a tumor suppressor gene on chromosome subband 10q23.3, is variably mutated and/or deleted in a variety of human cancers. Germline mutations in PTEN, which encode a dual-specificity phosphatase, have been implicated in at least two hamartoma tumor syndromes that exhibit some clinical overlap, Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome. Among several series of ovarian cancers, the frequency of loss of heterozygosity (LOH) of markers flanking and within PTEN, is approximately 30 to 50%, and the somatic intragenic PTEN mutation frequency is <10%. In this study, we screened primary adenocarcinomas of the ovary for LOH of polymorphic markers within and flanking the PTEN gene and for intragenic mutations of the PTEN gene and compared them to PTEN expression using immunohistochemistry. Furthermore, we sought to detect the expression of the presumed downstream targets of PTEN, such as P-Akt, p27, and cyclin D1 by immunohistochemistry. LOH at 10q23 was observed in 29 of 64 (45%) cases. Of the 117 samples, 6 somatic intragenic PTEN mutations, 1 germline mutation, and 1 novel polymorphism were found in 7 (6%) patients. Immunostaining of 49 ovarian cancer samples revealed that 13 (27%) were PTEN immunostain-negative, 25 (51%) had reduced staining, and the rest (22%) were PTEN expression-positive. Among the 44 informative tumors assessed for 10q23 LOH and PTEN immunostaining, there was an association between 10q23 LOH and decreased or absent staining (P = 0.0317). Of note, there were five (11%) tumors with neither mutation nor deletion that exhibited no PTEN expression and 10 (25%) others without mutation or deletion but had decreased PTEN expression. Among the 49 tumors available for immunohistochemistry, 28 (57%) showed P-Akt-positive staining, 24 (49%) had decreased p27 staining, and cyclin D1 was overexpressed in 35 (79%) cases. In general, P-Akt expression was inversely correlated with PTEN expression (P = 0.0083). These data suggest that disruption of PTEN by several mechanisms, allelic loss, intragenic mutation, or epigenetic silencing, all contribute to epithelial ovarian carcinogenesis, and that epigenetic silencing is a significant mechanism. The Akt pathway is prominently involved, but clearly not in all cases. Surprisingly, despite in vitro demonstration that p27 and cyclin D1 lies downstream of PTEN and Akt, there was no correlation between p27 and cyclin D1 expression and PTEN or P-Akt status. Thus, in vivo, although PTEN and Akt play a prominent role in ovarian carcinogenesis, p27 and cyclin D1 might not be the primary downstream targets. Alternatively, these observations could also suggest that pathways involving other than Akt, p27 and cyclin D1 that lie downstream of PTEN play roles in ovarian carcinogenesis.

Objectives: The goal of this study was to determine the maximally tolerated doses (MTDs) of carboplatin, paclitaxel (Taxol), etoposide, and cyclophosphamide (CTEC) with granulocyte-colony stimulating factor (G-CSF, Filgrastim) support as first-line chemotherapy in women with advanced epithelial ovarian cancer (EOC)., Methods: Newly diagnosed patients with either stage IV EOC, or stage III EOC and any amount of gross residual tumor after surgical debulking were eligible to receive six cycles of CTEC over five different dose levels in this phase I trial (planned 21-day cycle length). Paclitaxel, carboplatin, and cyclophosphamide were administered intravenously on Day 1, and oral etoposide was administered on Days 1, 2, and 3. G-CSF was administered beginning Day 4., Results: Twenty patients received a total of 98 cycles of CTEC over the five dose levels evaluated. Bone marrow suppression was the major toxic effect, with grade 4 neutropenia and thrombocytopenia being observed in 25 and 23% of cycles, respectively. The overall incidence of febrile neutropenia was 10%, and no toxic deaths occurred. No grade IV thrombocytopenia or febrile neutropenia was observed once the carboplatin dose was reduced from AUC of 7 to 5. Nonhematologic toxicity was generally mild (grade 2 or less). Dose-limiting toxicity was not observed at the highest dose level evaluated in this study, preventing assignment of the MTD. The clinical complete response rate was 92%, although 15 of 16 evaluable patients have progressed with a median progression-free interval of 4 months (range, 2-11 months). One patient remains disease-free 9 months from the completion of CTEC., Conclusions: The CTEC regimen is well tolerated and highly active. Although the MTD was not reached in this study, the short median progression-free interval suggests that this regimen is unlikely to be superior to standard treatment with paclitaxel and carboplatin. Strategies to optimize the development of future combination chemotherapy regimens in the treatment of newly diagnosed ovarian cancer are discussed., (Copyright 2000 Academic Press.)

Ovarian cancer cells frequently metastasize by implanting onto the peritoneal mesothelial surface of the abdominal cavity. Although the CD44 molecule expressed by ovarian cancer cells is known to partly mediate this process, the role of other adhesion proteins such as beta1-integrins has been previously difficult to demonstrate using the 4B4 anti-beta1 neutralizing antibody. In view of the widespread expression of beta1-integrins in ovarian cancer, however, we have further examined the potential role of this class of molecules in ovarian cancer cell implantation through the use of an alternative anti-beta1 neutralizing antibody, MAB13. We now report that MAB13 is capable of inhibiting the binding of three separate human ovarian cancer cell lines (36M2, CAOV-3, SKOV-3) to mesothelium (mean 37 +/- 4% inhibition, n = 21, P < 0.001). An additive inhibitory effect was observed when MAB13 was combined with anti-CD44 antibody (clone 515) (mean 63 +/- 3% inhibition, n = 19, P < 0.001), suggesting that binding occurs through two independent pathways involving both beta1-integrins and CD44. Because fibronectin is an extracellular matrix ligand recognized by many types of integrins and is abundantly expressed on mesothelial cells, the inhibitory effects of MAB13 and 4B4 on ovarian cancer cell binding to fibronectin were directly compared. MAB13 inhibited ovarian cancer cell binding to fibronectin to a significantly greater degree than did 4B4, suggesting that the discordant effects of these two antibodies on mesothelial adhesion may be partly related to their differential ability to neutralizing fibronectin-mediated binding. Studies using anti-alpha5 neutralizing antibody demonstrated that the alpha5beta1 fibronectin receptor contributes to approximately 50% of integrin-mediated binding of 36M2 and CAOV-3 cells (which express the alpha5 chain in 54 and 58% of cells, respectively). Since the RGD sequence of fibronectin is a known recognition site for many types of integrins, including alpha5beta1 and alphanubeta3, we performed binding in the continued presence of both anti-alpha5 and RGD peptide in order to exclude other types of fibronectin-integrin interactions. The addition of RGD peptides at concentrations known to be capable of blocking fibronectin binding resulted in no additional inhibitory effect over that observed with anti-alpha5 antibody alone, suggesting that alpha5beta1 was the major receptor responsible for fibronectin-mediated ovarian cancer binding to mesothelium. These data demonstrate that ovarian cancer cell binding to peritoneal mesothelium is mediated by several adhesion pathways and that simultaneous inhibition of both beta1-integrin and CD44 function may be necessary in order to significantly limit the intraabdominal spread of this tumor in vivo., (Copyright 1999 Academic Press.)

The BAX proapoptotic protein is capable of inducing cell death either directly, through its effects on mitochondrial function, or indirectly, by lowering the apoptotic threshold in response to certain chemotherapy agents. In this study, we tested the hypothesis that selective expression of BAX in human ovarian cancer through adenoviral gene transfer might represent a novel approach to eradicating tumor cells in vivo. Two constructs were prepared using replication-deficient adenoviral vectors containing either the cDNA for beta-galactosidase (Ad.DF3.betaGAL) or hemagglutinin (HA)-tagged BAX (Ad.DF3.BAX) under the control of the DF3 promoter. The DF3 promoter was used to confer tumor-specific gene expression in view of its restricted pattern of expression in the majority of human ovarian cancers and its limited expression in normal peritoneal mesothelial cells. In vitro infection of up to seven different epithelial cancer cell lines with Ad.DF3.betaGAL or Ad.DF3.BAX resulted in expression of either beta-galactosidase activity or HA-BAX protein, respectively, which was highly correlated with DF3 levels. Furthermore, infection with Ad.DF3.BAX was capable of highly selective cytotoxicity of DF3-positive ovarian cancer clonogenic cells in vitro. The effect of i.p. administration of Ad.DF3.BAX was also assessed in nude mice inoculated with the DF3-positive 36M2 human ovarian cancer cell line. Expression of either beta-galactosidase activity (after Ad.DF3.betaGAL treatment) or HA-BAX transcripts (after Ad.DF3.BAX treatment) was restricted to tumor tissue in vivo. Importantly, administration of Ad.DF3.BAX on days 2 and 3 after tumor inoculation was capable of eradicating >99% of tumor implants. These results demonstrate the feasibility of tumor selective expression of a proapoptotic protein such as BAX through adenoviral gene transfer.

Although paclitaxel is an important chemotherapy agent for the treatment of patients with epithelial ovarian cancer, its utility is significantly limited by the frequent development of drug resistance. Recent evidence suggests that resistance to chemotherapy may be partly related to defects in the apoptotic pathway. In this study we have investigated whether enhancement of apoptotic pathway function through stable expression of the BAD protein is capable of sensitizing human epithelial ovarian cancer cells to the effects of chemotherapy. Expression of HA-BAD in six separate clonal transfectants from two different ovarian cancer cell lines was found to significantly enhance the cytotoxic effects of paclitaxel, vincristine, and, to a lesser extent, etoposide. Enhancement of paclitaxel-induced apoptosis in HA-BAD-expressing clones was demonstrated by trypan blue exclusion, clonogenic cell assay, and flow cytometric evaluation. Importantly, this effect was associated with binding of HA-BAD to BCL-xL and concomitant disruption of BAX:BCL-xL interaction. Taken together, these data suggest that the development of small molecules which mimic the effects of BAD may represent a new class of drugs capable of preventing or reversing resistance to chemotherapy agents such as paclitaxel.

SW626 cells that overexpress BAX are sensitized to the cytotoxic effects of paclitaxel and vincristine. It has been assumed that BAX mediates these effects through its ability to alter mitochondrial function, specifically by promoting the release of cytochrome c and facilitating the mitochondrial permeability transition. However, we have found that several early paclitaxel-mediated events are enhanced in SW626 transfectants that overexpress BAX, including G2-M-phase arrest, tubulin polymerization, and BCL-2 phosphorylation. We now demonstrate that these seemingly disparate effects are explained by an enhanced accumulation of paclitaxel in BAX-overexpressing cells, an effect due to diminished drug efflux. In contrast, drug efflux is increased in cells that do not overexpress BAX, resulting in low intracellular paclitaxel levels and relative resistance to the effects of this drug. Drug efflux in SW626 cells is mediated by a verapamil-inhibitable, non-MDR-1, non-MRP-1 transporter whose function or expression may be inhibited by BAX. These data suggest that stable transfectants that overexpress BAX may be sensitized to apoptotic cell death through a novel mechanism involving the enhancement of intracellular levels of naturally occurring toxins such as alkaloid derivatives.

In addition to playing a role in tumorigenesis, loss of DNA mismatch repair results in low-level intrinsic resistance to cisplatin and carboplatin. We used a mismatch repair-deficient (clone B) and -proficient (clone B/rev) pair of Chinese hamster ovary sublines to determine the ability of cisplatin to enrich for repair-deficient cells during growth in vitro and in vivo. Clone B cells were 1.8-fold resistant to cisplatin as measured by a clonogenic assay. These cells were molecularly engineered to express constitutively the green fluorescent protein, and changes in the fraction of these repair-deficient cells were monitored by flow cytometric analysis. A single 1-hr exposure to cisplatin at an IC50 concentration enriched populations initially containing either 5 or 10% clone B cells by 81 and 75%, respectively, when measured at 5 days. Enrichment increased as a function of drug concentration to 158 and 169%, respectively, following an IC90 exposure. When grown as a xenograft, a single LD10 dose of cisplatin enriched the tumors by 48% from 4.6 to 6.8% repair-deficient cells (p = 0.04). To determine whether similar enrichment occurs during the treatment of human ovarian cancer patients, paired tumor samples were obtained from 38 patients before and after treatment with a minimum of 3 cycles of platinum drug-based primary chemotherapy and analyzed immunohistochemically for changes in the fraction of tumor cells expressing hMHL1. Following treatment there was a reduction in hMLH1 staining in 66% of the cases (p = 0.0005). Our results demonstrate that, despite the fact that loss of mismatch repair yields only modest levels of cisplatin resistance, even a single exposure to cisplatin produces quite a marked enrichment for repair-deficient cells in vitro and in vivo. Our results are consistent with the concept that treatment with cisplatin or carboplatin selects for preexisting mismatch repair-deficient cells, and that this contributes to the frequent development of clinical resistance.

Purpose: Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer., Methods: BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed., Results: BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified., Conclusion: The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

The p53 protein is known to play a central role in mediating G1 arrest or apoptosis in response to ionizing radiation in some cell types. It has been proposed that the link between p53 and induction of apoptosis is provided in part by p53-mediated upregulation of BAX. In this study, we used the human SW626 ovarian cancer cell line, which lacks functional p53, to further investigate the relationship between wildtype p53, BAX, and apoptosis. SW626 cells expressing a temperature sensitive (ts) p53 mutant did not undergo G1 arrest or apoptosis and did not exhibit enhanced sensitivity to radiation at the permissive temperature of 32 degrees C. The tsp53 protein was functional in these cells as evidenced by rapid induction of p21 at 32 degrees C, but not at 37 degrees C. Interestingly, restoration of wildtype p53 function at 32 degrees C was not associated with BAX upregulation. In addition, stable overexpression of BAX in SW626 cells was not capable of enhancing apoptotic cell death in response to radiation. Thus, failure of p53 to upregulate BAX is not the sole reason for its inability to promote radiation-induced apoptosis in SW626 cells. Taken together, our data suggest that neither p53 nor BAX upregulation is sufficient for the induction of apoptosis in response to genotoxic damage in some cell types.

Ovarian cancer cells frequently metastasize by implanting onto the peritoneal mesothelial lining of the abdominal cavity. Data obtained from in vitro adhesion studies have suggested a possible role for the CD44 molecule in this process. The purpose of the present study was to determine the in vivo role of CD44 in ovarian cancer metastasis by using a nude mouse xenograft model of peritoneal implantation. Three groups of 10 athymic female nude mice each received an i.p. inoculum of 10 x 10(6) cells from a CD44-positive human ovarian cancer cell line (36M2) in the presence of either anti-D144 antibody (Ab; nonreactive IgG1), anti-DF3 Ab (reactive IgG1 Ab that does not inhibit in vitro binding), or neutralizing anti-CD44 Ab (IgG1). The number of peritoneal and diaphragmatic implants at 5 weeks for anti-D144 and anti-DF3-treated groups was 103 +/- 17 and 120 +/- 20, respectively (mean +/- SE; P > 0.2). In contrast, animals treated with anti-CD44 Ab experienced a significant reduction in the number of tumor implants (35 +/- 4; P < 0.002). Anti-CD44 Ab was not inhibitory to the growth of 36M2 cells in vitro and did not inhibit s.c. tumor growth in vivo, suggesting that the observed effect was related to inhibition of peritoneal implantation. These data suggest that the CD44 molecule plays an important in vivo role in ovarian cancer cell implantation and that strategies to inhibit CD44 function may represent a novel approach to limiting the intra-abdominal spread of this highly lethal tumor.

To investigate the role of BAX in chemotherapy-induced apoptosis, we transfected the SW626 human ovarian cancer cell line, which lacks functional p53, with a cDNA encoding for murine BAX. Immunoblotting revealed that BAX transfectants expressed a mean of 10-fold increased levels of BAX compared with neo-transfected control clones, with similar levels of BCL-2 and BCL-xL. The cytotoxicity of paclitaxel, vincristine, and doxorubicin was significantly enhanced in BAX transfectants compared with control clones, whereas the cytotoxicity profile of carboplatin, etoposide, and hydroxyurea was unchanged. Increased paclitaxel-induced cytotoxicity of BAX clones was associated with enhanced apoptosis, as assessed by morphologic and flow cytometric criteria. These data suggest that sufficient levels of BAX may bypass the need for upstream molecules such as p53 in the process of chemotherapy-induced apoptosis.

Purpose: The presence of estrogen receptor (ER) and its therapeutic significance in ovarian borderline tumors (OBT) have not been established. We recently observed a response to tamoxifen therapy given empirically to a patient with unresectable, recurrent serous borderline tumor (SBT). In view of this observation the present study was undertaken to assess ER expression in 51 cases of OBT., Materials and Methods: ER expression was determined retrospectively, using an immunohistochemical method on formalin-fixed, paraffin-embedded specimens, from 35 cases of SBTs, 6 cases of mucinous mullerian (MMBT), and 10 cases of mucinous intestinal borderline tumors (MIBT). ER was considered positive if > 5% of tumor epithelial cell nuclei were immunostained. Both SBTs and mucinous borderline tumors (MBTs) were included to determine the influence of histologic type on ER expression., Results: The patients ranged in age from 25 to 77 years (median 43 years for SBTs, 36 years for MMBTs, and 37 years for MIBTs). The stage distribution for the SBTs was stage I in 27 patients (77%), stage II in 4 patients (11.5%), and stage III in 4 patients (11.5%). All patients with MBTs were stage I. ER expression was observed in the majority of cases and correlated with histologic type: 94% (33/35) of SBTs and 100% (6/6) of MMBTs were ER positive compared to 0% (0/10) of MIBTs (P < 0.01). In the SBT category the presence of ER did not correlate significantly with stage or age. In addition, ER was positive in all four SBT implants (including one involved lymph node) and two recurrent SBTs analyzed., Conclusion: ER expression is a common feature of SBT and MMBT, but not MIBT. The relevance of ER expression in the pathogenesis and treatment of OBTs requires further investigation.

Purpose: CD44 is a hyaluronic acid receptor that exists as a standard 90-kd form (CD44S) as well as several CD44 variant isoforms produced through alternative splicing. Expression of CD44 variants is associated with clinically aggressive behavior in some human tumors. The purpose of the present study is to define the expression of CD44 variant isoforms in ovarian cancer and to investigate whether the expression of these molecules is associated with adverse prognosis., Materials and Methods: Six specimens of normal ovarian surface epithelium (NOSE) and 31 separate cases of newly diagnosed ovarian cancer were studied by a combination of reverse-transcription polymerase chain reaction (RT-PCR) and immunoperoxidase staining. Clinical correlation was made between CD44 variant expression and stage (I/II v III/IV), residual disease (< or = 2.0- v > 2.0-cm mass), age (< or = 65 v > 65 years), histology (papillary serous v other), grade, and survival., Results: RT-PCR analysis revealed that NOSE predominantly expressed transcripts for CD44S, as well as a restricted pattern of transcripts characteristic of CD44 splice variants. CD44S and CD44 variant exon nine sequences (CD44-9v) were focally expressed in one of two NOSE specimens examined by immunoperoxidase staining. In comparison, the majority (71%) of ovarian cancer specimens expressed a complex pattern of CD44 splice variants by RT-PCR analysis. Immunoperoxidase studies revealed that the majority of ovarian cancer specimens expressed both CD44S and CD44-9v, whereas expression of sequences from variant exons 3, 4, and 6 was uncommon. There was no association between CD44 variant expression (transcript or protein) and stage, residual disease, age, histology, grade, or survival., Conclusion: Expression of CD44S and CD44-9v is a common feature of epithelial ovarian cancer cells. The lack of a significant association between CD44 variant expression and prognosis suggests that other factors may be more important in determining the clinical behavior of this disease.

Ovarian cancer cells disseminate by implanting onto the peritoneal mesothelial cell surface of the abdominal cavity. A common feature of these peritoneal implants is the presence of tumor cell invasion into the submesothelial extracellular matrix (ECM). In view of the important role of integrins in ECM recognition and cell migration, we were interested in defining the pattern of integrin expression and function in ovarian cancer cell lines and primary tissue samples. The beta 1 integrin chain was expressed by CAOV-3, SKOV-3, OVCAR-3, and SW626 ovarian cancer cell lines, associated with expression of alpha 1, -2, -3, -5, and -6 chains. The alpha 4 chain was also expressed by two of the four lines. In addition to beta 1 integrins, the alpha v beta 3 integrin was also expressed, although there was no expression of beta 2, -4, and -7 chains. Immunoprecipitation of surface-labeled CAOV-3 cells with an anti-beta 1 antibody revealed a band at approximately 110-130 kDa consistent with the known molecular mass of the beta 1 chain, as well as several associated bands consistent with noncovalently linked integrin alpha chains. A similar pattern of beta 1 and alpha v beta 3 integrin expression was observed for primary ovarian cancer tissue samples. Ovarian cancer cell lines exhibited significant binding to collagen type I and laminin which was primarily mediated by beta 1 integrins. In contrast, ovarian cancer cell binding to fibronectin was mediated by both alpha 5 beta 1 and alpha v beta 3 integrins. Even though mesothelial cells were observed to express fibronectin mRNA and protein, binding of ovarian cancer cells to peritoneal mesothelium was not blocked by neutralizing antibodies to beta 1 or alpha v beta 3 integrins. These data suggest that functional integrins are commonly expressed by ovarian cancer cells, although they do not appear to mediate ovarian cancer cell implantation onto peritoneal mesothelium. The role that integrins play in the invasion of ovarian cancer cells into the submesothelial ECM deserves further investigation.

We have previously shown that CD44 partly mediates ovarian cancer cell attachment to peritoneal mesothelium through recognition of mesothelial-associated hyaluronate. CD44 is a major receptor for hyaluronate and exists as a standard 90-180-kDa form (CD44H), as well as several higher molecular mass variant forms produced by alternative splicing. To determine whether functional differences exist between CD44H and its variants we have investigated the relationship between CD44 isoform expression and mesothelial adhesion in 12 ovarian cancer cell lines. Eight lines were CD44 positive (range, 83-94%) and demonstrated significant binding to mesothelium and hyaluronate, whereas two lines showed reduced CD44 levels (3-13%) and demonstrated decreased binding. Interestingly, two other lines (OVC-3 and SW626) expressed CD44 in the majority of cells (>93%) and yet bound weakly to mesothelium. Mean linear fluorescence intensity of CD44 expressed by OVC-3 and SW626 cells was approximately one-half that of strongly binding cell lines, suggesting that the ability to adhere may be partly related to CD44 surface density. However, immunoprecipitation and immunoblot analyses revealed that standard CD44H represented only 23-31% of total CD44 in weakly binding cells, with the majority of species being comprised of CD44 variants. Indirect immunofluorescence of OVC-3 and SW626 cells confirmed the presence of CD44 variants containing exons v3, v6, and v9. In contrast, CD44H represented the majority (75-86%) of total CD44 expressed by strongly binding cell lines such as CAOV-3 and UPN36T. Transfection of CD44H cDNA into weakly binding OVC-3 cells restored significant mesothelial binding which was partly blocked by anti-CD44 antibody. These data suggest that the expression of CD44 is necessary but not sufficient for mediating attachment of ovarian cancer cells to mesothelium. Although CD44 variants may constitute the major CD44 species in certain ovarian cancer cell lines, it appears that these CD44 species are not always capable of mediating significant binding to mesothelium or hyaluronate. Rather, an adequate level of CD44H is the critical determinant of binding in this system. The role of CD44 variants in the process of ovarian cancer metastasis will require further investigation.

Adhesion molecules such as selectins and integrins are known to mediate leukocyte attachment and transmigration through activated vascular endothelium. However, the molecules that mediate subsequent leukocyte entry into nonvascular spaces such as the abdominal cavity during states of peritoneal inflammation have not been identified. Because the peritoneal mesothelial lining represents the final barrier to leukocyte migration into the abdomen, it is likely that adhesion molecules expressed by mesothelial cells are involved in this process. We have developed an in vitro binding assay using confluent layers of normal human mesothelial cells to determine which adhesion molecules might be involved in T lymphocyte-mesothelial recognition. Normal peripheral blood T lymphocytes exhibit low-level specific binding to mesothelium (mean 13% specific binding, n = 4), which is enhanced by phorbol myristate acetate (PMA) treatment (mean 38% specific binding, n = 4). This binding is significantly inhibited in the combined presence of antibodies reactive with CD29 and CD18, suggesting a role for beta 1 and beta 2 integrins, respectively, in this interaction. Interestingly, cultured human mesothelial cells were shown to express vascular cell adhesion molecule-1 (VCAM-1), suggesting that this molecule might function as a counter-receptor for alpha 4 beta 1 expressed by T lymphocytes. Mesothelial cells were also noted to express ICAM-1, CD29, and CD44, but not CD18 or selectins. VCAM-1 expression was not a constitutive property of freshly obtained mesothelial cells but was inducible upon culture in the presence of either interleukin-1 (IL-1), tumor necrosis factor (TNF), or PMA. Neutralizing antibodies reactive with either alpha 4, VCAM-1, or CD29 were all equally capable of inhibiting the binding of activated leukocytes to mesothelial cells (in the presence of anti-CD18 antibody). Mesothelial VCAM-1 was found to have a molecular mass of 110 kD and an mRNA transcript of approximately 3.2 kb, consistent with the predominant VCAM-1 species found in activated endothelium. These data suggest that functional VCAM-1 is expressed on activated mesothelial cells and may play a role in the distal arm of leukocyte trafficking to the abdominal cavity.

Epithelial cancer of the ovary spreads by implantation of tumor cells onto the mesothelial lining of the peritoneal cavity. We have developed an in vitro binding assay using confluent monolayers of normal peritoneal mesothelial cells in order to assess the role of known adhesion proteins in this process. Cells from normal ovarian surface epithelium and the ovarian cancer cell lines CAOV-3 and SKOV-3 exhibited significant adhesion to mesothelium in vitro (range 33-56% specific binding). Although these cells expressed several adhesion molecules, including CD44 and integrins such as alpha 4 beta 1, alpha 5 beta 1, and alpha v beta 3, only anti-CD44 antibody was capable of inhibiting mesothelial binding (range 42-44% inhibition). Adhesion molecule expression was also determined for fresh ovarian specimens, with CD44 being expressed in 2 of 2 cases of normal ovarian epithelium, 15 of 16 (94%) cases of tissue-derived tumor (from primary sites or peritoneal implants), and only 2 of 8 (25%) cases of free-floating tumor cells from ascites. Three of three CD44-positive cases derived from peritoneal implants exhibited significant mesothelial binding which was partly blocked by anti-CD44 antibody, whereas 2 of 2 CD44-negative cases derived from ascites showed minimal binding. CD44-mediated binding of ovarian cancer cells was determined to be due to recognition of mesothelium-associated hyaluronate, suggesting that the CD44H isoform was involved in this process. Immunoprecipitation of the CD44 species expressed by ovarian cancer cells revealed 2 major bands at 85-90 and 180 kDa, consistent with the known molecular masses of CD44H. These results suggest that CD44H may be an important mediator of ovarian cancer cell implantation and that decreased CD44H expression may be associated with release of cells into the peritoneal space during ascites formation. It is possible that strategies to interfere with CD44H function may result in decreased intraabdominal spread of this highly lethal neoplasm.

Purpose: Attempts to increase dose-intensity in clinical practice have been limited by cumulative hematologic toxicity despite the use of hematopoietic growth factors. To address this problem, we designed a study to determine whether four cycles of dose-intensive chemotherapy with carboplatin could be administered in the outpatient setting using granulocyte-macrophage colony-stimulating factor (GM-CSF) and peripheral-blood progenitor cells (PBPCs) that had been harvested before initiation of treatment., Patients and Methods: An initial cycle (cycle no. 0) of cyclophosphamide 4 g/m2 followed by GM-CSF was used to mobilize PBPCs harvested by leukapheresis for 6 consecutive days. Cycles no. 1 through 4 consisted of outpatient carboplatin 600 mg/m2 and cyclophosphamide 600 mg/m2 followed by GM-CSF 5 micrograms/kg subcutaneously (SC) twice per day every 28 days. In cycle no. 1, PBPC were not reinfused to assess the effects of GM-CSF alone. In cycles no. 2 through 4, PBPCs were reinfused on day 3 in an outpatient setting., Results: In eight assessable patients, the addition of PBPCs in cycle no. 2 resulted in a significant reduction in the median duration of thrombocytopenia less than 20,000/microL (6.5 v 1 day; P = .016), days to platelets more than 50,000/microL (20.5 v 15 days; P = .020), number of platelet transfusions (five v 1.5; P = .016), and duration of neutropenia (absolute neutrophil count [ANC] < 1,000/microL (7 v 2.5 days; P = .008) when compared with cycle no. 1. Dose-limiting hematologic toxicity, defined as more than 7 days of platelets less than 20,000/microL or ANC less than 500/microL, was observed in four of eight patients during cycle no. 1, but not during cycles no. 2, 3, and 4 of chemotherapy supported by PBPCs (a total of 19 cycles in eight patients). Five of eight patients completed all four cycles of high-dose therapy. Three patients did not complete four cycles due to late thrombocytopenia (n = 2) or tumor progression (n = 1)., Conclusion: These results indicate a benefit of PBPCs in addition to GM-CSF in alleviating myelosuppression of dose-intensive chemotherapy. Initial collection of PBPCs may allow administration of repetitive cycles of high-dose chemotherapy with acceptable toxicity to outpatients at disease onset.

Prolonged exposure to low concentrations of cytarabine preferentially inhibits in vitro growth of neoplastic myeloid progenitors from patients with chronic myelogenous leukemia (CML) compared to that of normal myeloid progenitors. Continuous infusions of cytarabine in doses of 15-30 mg/m2/day were therefore administered for extended periods to patients with CML in stable phase to determine if this treatment could achieve selective cytoreduction of Philadelphia chromosome (Ph)-positive cells. Five patients demonstrating > 90% Ph-positive metaphases before treatment received a total of 43 cycles of cytarabine infusional therapy. Cytarabine was administered on an outpatient basis using a portable, battery-operated syringe pump until the total leukocyte count reached 2500/microliters or the platelet count reached 75,000/microliters. A new cycle was begun when the total leukocyte count exceeded 4,000/microliters and the platelet count exceeded 100,000/microliters. The median duration of cytarabine administration per cycle was 29 days (range 15-72 days). Leukocytosis was readily controlled by low-dose cytarabine therapy in all patients. All five patients experienced complete hematologic responses during cytarabine therapy. The fraction of Ph-positive metaphases in the marrow of the five patients was reduced to 0, 10%, 43%, 72%, and 84%, respectively, during therapy. The median time to achieve optimal cytogenetic response was 4.8 months (range 2.8-8.6 months). One patient demonstrated a complete cytogenetic response after three cycles of cytarabine. Another patient demonstrated persistent cytogenetic improvement during 20 cycles of cytarabine, with a median 38% Ph-positive marrow metaphases (range 10-53%) over 32 months. Cytarabine therapy was generally well-tolerated, but was discontinued in one patient because of persistent asymptomatic elevations in hepatic enzymes, which resolved within 2 months after discontinuing therapy. There were no episodes of fever during neutropenia, and platelet transfusions were not required. However, symptomatic anemia requiring transfusion of red cells occurred during most cycles of treatment. In summary, treatment of CML with low-dose cytarabine can induce prolonged cytogenetic improvement in some patients with acceptable toxicity. Further evaluation is needed to ascertain the effect of this treatment on duration of stable phase and overall survival.

The treatment of patients with relapsed or refractory acute myeloid leukemia (AML) with high dose cytosine arabinoside (ara-C) results in short-lived complete response rates of 30-50%. We have previously shown that entry of myeloid leukemic cells into S phase can be accelerated in vitro through the use of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), resulting in enhancement of ara-C-mediated cytotoxicity. In order to evaluate the in vivo biological and clinical effects of this strategy in patients with high risk AML, we treated three patients with either refractory or relapsed disease with a continuous infusion of rhGM-CSF (0.45 micrograms/kg/h aglycoprotein) for 18 h, followed by the institution of high dose ara-C and continuation of rhGM-CSF throughout the 4 day duration of ara-C treatment. Prior to therapy, no patient had detectable levels of circulating rhGM-CSF, and there was no evidence of GM-CSF receptor occupancy in leukemic myeloblasts. After 18 h of rhGM-CSF therapy, all patients had biologically active levels of circulating rhGM-CSF (7.9-12.0 ng/ml), and two patients showed a significant degree of leukemic GM-CSF receptor occupancy without evidence of GM-CSF receptor down-regulation. A significant rise in the S phase fraction of leukemic myeloblasts was observed at 18 h of rhGM-CSF treatment in all three patients (29-56% increment). The toxicity of combined rhGM-CSF/ara-C therapy included pericarditis and cerebellar degeneration in one patient, fever and mild renal dysfunction in two patients, and mild hepatic dysfunction in all three patients. Each patient showed a transient rise in the absolute neutrophil and blast count during rhGM-CSF/ara-C administration, followed by profound, but clinically tolerable, myelosuppression. No patient developed clinical evidence of leukostasis. There was one death related to pericardial tamponade, one death related to refractory disease, and one clinical and cytogenetic remission. These results suggest that exogenously administered rhGM-CSF is capable of rapidly mobilizing leukemic cells into S phase in vivo and theoretically should be useful in overcoming kinetic resistance to ara-C. Clinical trials of this regimen in patients with high risk AML who are not already pharmacologically resistant to ara-C are warranted.

Myeloid leukemic progenitor cells proliferate in vitro in response to a variety of humoral factors, the most prominent among these being granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanism by which GM-CSF transduces its proliferative signal in acute myeloid leukemia has been extensively investigated over the past year. It is now known that the GM-CSF belongs to a new family of hematopoietic growth factor binding proteins which are characterized by a relatively short intracytoplasmic domain that lacks a tyrosine kinase sequence. Nevertheless, studies performed using GM-CSF-dependent leukemic cell lines demonstrate the appearance of several new phosphotyrosine species after GM-CSF exposure, suggesting that receptor activation is directly or indirectly linked to tyrosine kinase stimulation. Apart from the basic biology of GM-CSF-induced signal transduction, the ability of this factor to enhance the S-phase fraction of myeloid leukemia blasts may have important therapeutic implications. Clinical trials are currently being conducted in an attempt to determine whether GM-CSF is able to overcome kinetic resistance of leukemic myeloblasts to cell cycle-specific agents such as cytarabine in the treatment of patients with acute myeloid leukemia.

The human myeloid cell line MO7 requires either granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) for proliferation. We have previously shown that both GM-CSF and IL-3 transiently induce tyrosine phosphorylation of a number of proteins, including two cytosolic proteins, p93 and p70, which are maximally phosphorylated 5-15 min after addition of growth factor to factor-deprived cells. GM-CSF-induced proliferation of MO7 cells was found to be inhibited by two activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA) and bryostatin-1. PMA did not affect surface expression or affinity of the GM-CSF receptor but significantly inhibited GM-CSF- or IL-3-induced tyrosine phosphorylation of p93 and p70. In contrast, PMA augmented GM-CSF-induced tyrosine phosphorylation of another protein, p42. Pretreatment of cells with sodium orthovanadate to inhibit protein tyrosine phosphatases (PTPase) partially reversed the inhibitory effects of PMA. These results suggest that one aspect of GM-CSF and IL-3 signal transduction, protein tyrosine phosphorylation, can be inhibited by a mechanism which does not involve receptor down-regulation, and may involve either receptor down-regulation, and may involve either inhibition of a receptor-activated tyrosine kinase, activation of a protein tyrosine phosphatase, or both. This mechanism could be important in exerting control of proliferation of some types of hematopoietic cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates a broad range of myeloid cells through binding to high affinity surface membrane receptors. The effects of this hematopoietin are dependent upon the differentiation status of the myeloid cell and range from proliferation of early myeloid progenitor cells to activation of neutrophil and monocyte function. In addition, many of the biological effects of GM-CSF are shared with interleukin-3 (IL-3), a distantly related lymphokine. In this study, we have characterized the GM-CSF receptor of myeloid cells at various stages of differentiation by comparing the binding characteristics and surface regulation of this receptor in early versus late myeloid cells. Scatchard analysis revealed a single class of high affinity receptors on normal neutrophils, monocytes, and myeloblasts from patients with acute myeloid leukemia. Neutrophils expressed significantly higher numbers of receptors, with an approximately 2-fold lower affinity, when compared with other myeloid cells. Two different patterns of GM-CSF receptor regulation and binding were observed. In the first pattern, the GM-CSF receptor of neutrophils was rapidly down-regulated by GM-CSF itself, by phorbol myristate acetate (PMA), and by the calcium ionophore A23187, and it was not competed for by IL-3 (class I receptor). In contrast to the neutrophil receptor, the GM-CSF receptor of the myeloblast demonstrated resistance to the down-regulatory effects of GM-CSF itself, PMA, and A23187, and it was completely competed for by IL-3 (class II receptor). In some cases of acute myeloid leukemia and monocytes, a mixed pattern of partial PMA responsiveness and partial competition by unlabeled IL-3 was observed, suggesting the coexpression of both class I and II receptors in these cells. In these cells, after down-regulation of the class I receptor by PMA, the remaining receptors were shown to be completely cross-competed for by IL-3, further supporting the hypothesis that these cells have a mixture of class I and II receptors. Chemical cross-linking of radiolabeled GM-CSF to myeloid cells revealed the labeling of three proteins (156, 126, and 82 kDa) which were identical in cells expressing either class I or II binding sites. These data show that there are differentiation-associated differences in the regulation of the GM-CSF receptor which may have important physiological consequences.

CML has provided a model for the genetic basis of human neoplasia. Since 1960 with the discovery of the Ph chromosome, study of this disease has provided a conceptual basis for viewing cancer as a clonal disorder occurring at the stem cell level and associated with intrinsic genetic defects which contribute to abnormal growth regulation. Although several oncogenes have been identified through the study of tumor-producing retroviruses in animals, discovery of the BCR/ABL translocation, the altered 8.5 kb BCR/ABL transcript, and the hybrid BCR/ABL P210 protein with enhanced tyrosine kinase activity has provided one of the first examples of a human neoplasm in which structural alterations in a normal cellular gene might lead to malignant transformation. However, it is likely that P210 is necessary but not sufficient for the full spectrum of malignant behavior observed in this disease. Investigation of the molecular events that are associated with the additional cytogenetic abnormalities of blast phase will most likely reveal alterations of other important growth regulatory genes which contribute to the multistep nature of malignant transformation in CML.

Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors (Kd = 30-100 pM). By using radiolabeled GM-CSF with high specific activity, we have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations (95% +/- 1%, 34% +/- 5%, 48% +/- 8% receptor down-regulation, respectively). GM-CSF also down-regulated its own receptor on monocytes and myeloid progenitor cells. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, we also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.