Repair of DNA following incorporation of 1-beta-D-arabinofuranosylcytosine into herpes simplex virus type 1.

The nucleoside analogue 1-beta-D-arabinofuranosylcytosine (ara-C) is incorporated into herpes simplex virus type 1 (HSV-1) DNA, and this correlates with inhibition of virus replication. The technique of Weigle-type reactivation (WR) was used to compare the ability of induced cellular DNA repair pathways to recognize or repair ara-C incorporated into HSV-1 DNA and ultraviolet (UV)-irradiated virus DNA (254 nm). Pretreatment of monkey cells with low-fluence UV irradiation, growth in cis-dichlorodiammineplatinum(II), or growth in ara-C followed by infection after a 24-hr incubation period resulted in enhanced survival of UV-irradiated HSV-1. Under the same experimental conditions, no reactivation of HSV-1 inactivated by growth in ara-C is observed. Comparisons between uninfected Vero cells exposed to UV irradiation (30 J/m2) or grown in 10(-6) M ara-C demonstrated repair replication in irradiated cells, whereas there was no evidence for DNA repair at various time intervals following removal of the nucleoside analogue. These observations suggest that, once ara-C is incorporated into HSV-1 or eukaryotic DNA, it is not recognized as a repairable lesion within the limits of the DNA repair assays used in these studies.

Detection of sequence-specific antitumor alkylating agent DNA damage from cells treated in culture and from a patient.

Detection of sequence-specific DNA damage induced by antitumor alkylating agents might provide a mechanism for detecting and discriminating damage specific to one or more of these drugs. Using repetitive primer-extension and human alphoid DNA as a substrate, lesions specific for an activated form of cyclophosphamide, 4-hydroperoxycyclophosphamide, were detected at 32 of 33 guanines within a 200-base pair region in DNA from cells treated in culture. There was a marked variation in lesion site intensity among affected guanines. For instance, guanines flanked by cytosine were weak sites of 4-hydroperoxycyclophosphamide-induced damage. Damage at bases other than guanine induced by cisplatin, UV irradiation, and adozelesin were compared to drug-DNA lesions induced by 4-hydroperoxycyclophosphamide. Using this method it was possible to detect, and at some sites distinguish, between cyclophosphamide- and cisplatin-induced DNA damage within WBC DNA from a patient treated with both agents. There was a different damage pattern for DNA derived from cells treated in culture compared to DNA derived from the patient sample.

Regional loss of chromosome 6 in two urological malignancies.

Immunogenetic evidence suggests a genetic association between the major histocompatibility complex and the two genitourinary neoplasms, testicular teratocarcinoma and renal cell carcinoma. In order to develop a possible explanation for these findings, we designed a series of experiments to investigate the existence of a tumor suppressor gene in the region of HLA by looking for loss of germ line heterozygosity in these neoplasms at loci within and centromeric to HLA on chromosome 6. Restriction enzyme-digested DNA, from 15 human teratocarcinoma tumors, and corresponding normal somatic DNA, from peripheral blood mononuclear cells, were hybridized to one of three chromosome 6 probes determined to be polymorphic in this region. Probe 4c11 (6p11-cen) revealed loss of germ line DNA in three of 14 tumors. In contrast, probes pC22A (6p21.3) and p308 (6cen), which hybridize to chromosome 6p sequences, telomeric and centromeric to those sequences recognized by 4c11, did not demonstrate loss or sequence alteration in a total of 14 analyzable tumors. A total of 33 renal cell carcinoma specimens was also analyzed with the informative 4c11 probe with loss demonstrated in six of 33 tumors. In contrast, 23 different samples representing 15 other tumor types were examined with 4c11. Loss of chromosome 6p DNA was demonstrated in only two samples. These data support the hypothesis that there is nonrandom loss of DNA centromeric to HLA on chromosome 6 in both testicular teratocarcinoma and renal cell carcinoma.

Selection for androgen receptor mutations in prostate cancers treated with androgen antagonist.

The role of androgen receptor (AR) mutations in androgen-independent prostate cancer (PCa) was determined by examining AR transcripts and genes from a large series of bone marrow metastases. Mutations were found in 5 of 16 patients who received combined androgen blockade with the AR antagonist flutamide, and these mutant ARs were strongly stimulated by flutamide. In contrast, the single mutant AR found among 17 patients treated with androgen ablation monotherapy was not flutamide stimulated. Patients with flutamide-stimulated AR mutations responded to subsequent treatment with bicalutamide, an AR antagonist that blocks the mutant ARs. These findings demonstrate that AR mutations occur in response to strong selective pressure from flutamide treatment.

Cyclophosphamide, carmustine, and etoposide with autologous bone marrow transplantation in refractory Hodgkin's disease and non-Hodgkin's lymphoma: a dose-finding study.

Cyclophosphamide, carmustine (BCNU), and etoposide (VP-16) (CBV) is a widely used conditioning regimen in autologous bone marrow transplantation (ABMT) of patients with refractory and relapsed lymphoma. However, the maximum-tolerated dose (MTD) of these agents when used in combination has not been systematically explored. We treated 58 patients (28 with non-Hodgkin's lymphoma [NHL], 30 with Hodgkin's disease [HD]) at seven dose levels of CBV. Doses were cyclophosphamide 4,500 to 7,200 mg/m2, BCNU 450 to 600 g/m2, and VP-16 1,200 to 2,000 mg/m2. The MTD was cyclophosphamide 7,200 mg/m2, BCNU 450 mg/m2, and VP-16 2,000 mg/m2. Six hundred milligrams per square meter of BCNU was associated with five of 18 cases of interstitial pneumonitis versus two of 40 at 450 mg/m2 (P = .02). Treatment-related mortality was 5% at dose levels less than or equal to the MTD and 22% at the highest dose. In this heavily pretreated patient population, most of whom had high volume residual disease, complete responses (CRs) to CBV and ABMT occurred in 25% of assessable patients with NHL and 43% of patients with HD. Thirteen of 28 patients with NHL and 14 of 30 with HD remain free from disease progression with median follow-up of 212 and 215 days, respectively. CBV can be administered with acceptable toxicity over a wide range of doses to patients with refractory and relapsed lymphoma.

Eligibility and response guidelines for phase II clinical trials in androgen-independent prostate cancer: recommendations from the Prostate-Specific Antigen Working Group.

PURPOSE: Prostate-specific antigen (PSA) is a glycoprotein that is found almost exclusively in normal and neoplastic prostate cells. For patients with metastatic disease, changes in PSA will often antedate changes in bone scan. Furthermore, many but not all investigators have observed an association between a decline in PSA levels of 50% or greater and survival. Since the majority of phase II clinical trials for patients with androgen-independent prostate cancer (AIPC) have used PSA as a marker, we believed it was important for investigators to agree on definitions and values for a minimum set of parameters for eligibility and PSA declines and to develop a common approach to outcome analysis and reporting. We held a consensus conference with 26 leading investigators in the field of AIPC to define these parameters. RESULT: We defined four patient groups: (1) progressive measurable disease, (2) progressive bone metastasis, (3) stable metastases and a rising PSA, and (4) rising PSA and no other evidence of metastatic disease. The purpose of determining the number of patients whose PSA level drops in a phase II trial of AIPC is to guide the selection of agents for further testing and phase III trials. We propose that investigators report at a minimum a PSA decline of at least 50% and this must be confirmed by a second PSA value 4 or more weeks later. Patients may not demonstrate clinical or radiographic evidence of disease progression during this time period. Some investigators may want to report additional measures of PSA changes (ie, 75% decline, 90% decline). Response duration and the time to PSA progression may also be important clinical end point. CONCLUSION: Through this consensus conference, we believe we have developed practical guidelines for using PSA as a measurement of outcome. Furthermore, the use of common standards is important as we determine which agents should progress to randomized trials which will use survival as an end point.

A multi-institutional phase ii study of SU101, a platelet-derived growth factor receptor inhibitor, for patients with hormone-refractory prostate cancer.

In a multi-institutional Phase II trial, we evaluated the efficacy of a platelet-derived growth factor receptor (PDGF-r) inhibitor, SU101, in patients with hormonerefractory prostate cancer. The patients received a 4-day i.v. loading dose of SU101 at 400 mg/m(2) for 4 consecutive days, followed by 10 weekly infusions at 400 mg/m(2). The primary study end points were a decline in prostate-specific antigen (PSA) and a decrease in measurable tumor. Secondary end points were time to progression and an effect on pain as measured by the Brief Pain Survey. Expression of PDGF-r was examined in both metastatic and archival primary prostate tumor samples. Forty-four patients were enrolled at four centers. The median age was 72 years, the median PSA was 223 ng/ml, and 21 patients had at least one prior chemotherapy. Thirty-nine patients are evaluable for PSA, and three patients demonstrated a PSA decline >50% from baseline (55-99.9% decrease). The median time to progression was 90 days. Of 19 patients evaluable for measurable disease, 1 patient had a partial response. Nine of 35 evaluable patients had significant improvement in pain. The most frequent adverse events were asthenia (75%), nausea (55%), anorexia (50%), and anemia (41%). PDGF-r expression was detected in 80% of the metastases and 88% of primary prostate cancers. The results of this trial may warrant further clinical studies with other PDGF-r inhibitors.

Functional characterization of mutant androgen receptors from androgen-independent prostate cancer.

Mutations in the androgen receptor (AR), that alter steroid hormone specificity have been identified in a series of androgen-independent prostate cancers. To address the functional properties of these mutant ARs that may have contributed to their selection in vivo, responses to a series of steroid hormones and antiandrogens were assessed. CV-1 cells were cotransfected with wild-type or mutant ARs and a luciferase reporter plasmid regulated by an androgen-responsive element. Dose-response curves were analyzed for 5alpha-dihydrotestosterone, the most active androgen in normal prostate, and androstenedione, a major androgen derived from the adrenals. Although the mutant ARs responded to both of these steroids, the responses were equivalent to or less than the wild-type AR. In contrast, responses to flutamide, a competitive antagonist of the wild-type AR, were markedly increased by three of the mutations. Similar responses were observed with a second antiandrogen, nilutamide. Bicalutamide, another antiandrogen related to flutamide, remained an antagonist for these mutant ARs. Finally, flutamide was observed to be a weak partial agonist of the wild-type AR in this system. These results indicate that flutamide used in conjunction with androgen ablation therapy for prostate cancer may select for tumor cells with flutamide-inducible ARs.

A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer.

A recombinant vaccinia virus encoding human prostate-specific antigen (rV-PSA) was administered as three consecutive monthly doses to 33 men with rising PSA levels after radical prostatectomy, radiation therapy, both, or metastatic disease at presentation. Dose levels were 2.65 x 10(6), 2.65 x 10(7), and 2.65 x 10(8) plaque forming units. Ten patients who received the highest dose also received 250 microg/m2 granulocyte-macrophage colony-stimulating factor (GM-CSF) as an immunostimulatory adjunct. No patient experienced any virus-related effects beyond grade I cutaneous toxicity. Pustule formation and/or erythema occurred after the first dose in all 27 men who received > or =2.65 x 10(7) plaque forming units. GM-CSF administration was associated with fevers and myalgias of grade 2 or lower in 9 of 10 patients. PSA levels in 14 of 33 men treated with rV-PSA with or without GM-CSF were stable for at least 6 months after primary immunization. Nine patients remained stable for 11-25 months; six of these remain progression free with stable PSA levels. Immunological studies demonstrated a specific T-cell response to PSA-3, a 9-mer peptide derived from PSA. rV-PSA is safe and can elicit clinical and immune responses, and certain patients remain without evidence of clinical progression for up to 21 months or longer.

Docetaxel, estramustine, and short-term androgen withdrawal for patients with biochemical failure after definitive local therapy for prostate cancer.

Over the past 10 years, men with prostate cancer have received earlier diagnoses and are undergoing prostatectomy and/or radiation therapy with curative intent; however, many men have increasing prostate-specific antigen (PSA) levels without evidence of local progression or metastatic disease during the first 2 years after definitive local therapy. Optimal treatment of men with PSA-only recurrent prostate cancer has not been established. This ongoing phase II trial is evaluating docetaxel (70 mg/m(2) administered intravenously over 1 hour on day 2 every 21 days for four cycles) and estramustine (10 mg/kg/d orally on days 1 to 5 every 21 days for four cycles) followed by bicalutamide and goserelin acetate in men with increasing PSA levels after prostatectomy and/or radiation therapy. Patients received pretreatment with dexamethasone, and after the third patient enrolled, patients received warfarin for prophylaxis against thrombosis. Colony-stimulating factor support was allowed. In preliminary results, 11 of 15 patients completed protocol chemotherapy; 12 of 15 patients achieved complete response (ie, normalization of PSA) after four cycles of chemotherapy. In addition, testosterone levels were reduced to the castrate range in all patients after chemotherapy. The regimen was generally well tolerated, and toxicities were mostly hematologic, with grade (3/4) neutropenia reported in approximately half of patients. Preliminary results of this phase II trial are encouraging, and enrollment is ongoing.

Differences in in vivo and in vitro sequence-specific sites of cisplatin-DNA adduct formation and detection of a dose-response relationship.

The cytotoxic and mutagenic properties of the anticancer drug cis-diammine-dichloroplatinum(II) (cisplatin) are mediated by bifunctional adducts between purines. Experiments performed in this study employed a new repetitive thermal-cycling technique to detect cisplatin adduct formation following exposure of cells in culture (in vivo) or following treatment of purified DNA (in vitro exposure). The initial goal of this study was to determine if cisplatin-DNA adduct formation could be measured accurately using phosphor-imaging over a broad concentration range. If this proved possible, it would then be feasible to determine if adduct formation differed within chromatin compared with purified DNA. There were no significant differences in the cisplatin-DNA adduct pattern induced in closed circular or linear double-stranded plasmids in vitro, suggesting that this type of tertiary structural change does not affect the formation of adduct sites. Sequence-specific DNA adduct formation within a human repetitive DNA target sequence, alphoid DNA, following cisplatin treatment of prostate cancer cells in culture (in vivo) and treatment of purified DNA in vitro revealed consistent increases in adduct formation over a broad concentration range, validating the experimental technique. Comparing preferences for cisplatin adduct site formation under these different conditions of exposure demonstrated statistically significant differences. Similar differences were detected for cisplatin repair-deficient Xeroderma pigmentosum cells treated in cell culture, indicating that in vivo/in vitro preferences for adduct site formation are not the result of DNA repair in vivo.

Effect of DNA conformation on cisplatin adduct formation.

The anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) has been shown previously to form adducts preferentially within internucleosomal or linker DNA rather than to DNA within the nucleosome. To determine whether other "open" regions of chromatin have an increased affinity for cisplatin, adduct formation within specific chromatin domains was analyzed. There was a significant increase in cisplatin-DNA adduct formation for DNA associated with the nuclear matrix (NM) compared with other chromatin domains and total unfractionated DNA. In contrast, treatment of the same cells with trans-diamminedichloroplatinum(II) (transplatin) did not result in preferential adduct formation. These findings led to the hypothesis that it might be possible to alter DNA to make it a more favorable target for cisplatin. The effect of arginine butyrate on cisplatin-DNA adduct formation was analyzed in human cancer cells. The combination of arginine butyrate and cisplatin resulted in a concentration-responsive increase in cisplatin-DNA adduct formation in PC-3 cells and an overall increase in cisplatin-DNA adduct formation in three other human cancer cell lines. The same combination also resulted in a significant increase in drug-induced cytotoxicity at a low concentration of cisplatin. These results suggest that chromatin configuration can affect cisplatin adduct formation.

Treatment of metastatic prostate cancer. Lessons from the androgen receptor.

The exquisite hormonal dependence of prostate cancer continues to provide an opportunity and a challenge for oncologists. It is clear that future efforts in the laboratory should include determining the frequency and spectrum of AR mutations in AI prostate cancer, the development of more effective antiandrogens, and understanding in greater detail how the AR stimulates the growth of prostate cancers. These efforts may eventually lead to treatments that greatly reduce any stimulatory effects of the AR on prostate cells, possibly resulting in a significant improvement in disease-free survival and, perhaps in conjunction with other modalities, cure of some earlier stages of disease. And even for patients with advanced disease, because hormonal therapy is generally fairly well tolerated even in the typically older prostate cancer patient, defining the contribution of AR-mediated growth to AI disease will be critically important.

Prostate cancer in the older man.

Most men diagnosed with prostate cancer are more than 65 years of age. Therefore, a discussion of the issues surrounding the diagnosis, prevention, and treatment of prostate cancer in older men is, in many ways, a review of prostate cancer in general. Nonetheless, older patients with prostate cancer are often faced with a different set of problems than younger patients. For instance, if preventive strategies prove useful, they will have important implications for older men. Even a significant delay in diagnosis could greatly benefit the elderly population. Prostate-specific antigen (PSA) screening is controversial for men of any age but, for older men, screening may impart a risk to quality of life that may outweigh the potential advantages of diagnosis and treatment. Results of a large follow-up study of patients treated with radical prostatectomy suggest that even men with rising PSA values after surgery can have a relatively benign and protracted course. The survival rates noted in this study, however, were only for a select population of surgical patients, and, in fact, higher prostate cancer death rates have been observed for patients adopting the watchful waiting approach. Older men who request some form of primary therapy are increasingly being treated with brachytherapy, despite the lack of randomized trials demonstrating efficacy compared to external-beam radiation therapy, surgery, or watchful waiting. Contrary to an often-held view, older prostate cancer patients may have more morbidity from long-term testosterone suppression than younger patients. On the other hand, chemotherapy seems to be as well tolerated overall in older patients as in younger patients.

Is the flare phenomenon clinically significant?

OBJECTIVES: The existing luteinizing hormone-releasing hormone (LHRH) analogs have been the preferred method of inducing androgen deprivation for prostate cancer for over a decade. These agents are well known to cause a surge in serum testosterone levels during the first week of therapy. However, there are wide discrepancies in reports of the frequency and severity of acute clinical progression or clinical flare that might result from the testosterone surge. Also, there is not a clear consensus as to whether antiandrogens should be routinely given to all patients during the first month of LHRH therapy to prevent flare responses. METHODS: Clinical trials involving LHRH analog therapy for prostate cancer were reviewed, and the frequency of clinical flare responses noted. Particular attention was given to the kinds of clinical problems associated with the flare response. The use of LHRH analog therapy in treatment of patients with prostate cancer for indications other than overt metastatic disease is discussed, because this is becoming a much more common use of these agents. This article analyzes 2 placebo-controlled, double-blind trials testing the effectiveness of existing antiandrogens in ameliorating flare responses. RESULTS: The use of LHRH analogs for patients with stage D2 disease can be associated with clinical flare in approximately 10% of D2 patients. In addition to bone pain, cord compression, and bladder outlet obstruction, another potentially severe side effect is cardiovascular risk arising presumably from hypercoagulability associated with a rapid increase in tumor burden. In clinical series involving D2 patients, the frequency of clinical flare greatly varies, probably because of the level of scrutiny of the investigator and/or the prostate-cancer tumor burden present at the initiation of therapy. Concomitant antiandrogen therapy reduces, but does not totally eliminate, the flare responses in patients at high risk for flare. Treating prostate cancer in the D0 stage or in the neoadjuvant setting will result in biochemical evidence of testosterone surge, but these patients are at very little risk for clinical flare responses. CONCLUSIONS: There is a wide variation in the reported frequency of clinical flare responses from LHRH analogs during the initial treatment of patients with stage D2 disease. The risk-to-benefit ratio, especially in patients with symptomatic bone metastasis, would dictate routine use of antiandrogen therapy for the first month of LHRH analog treatment. For patients at risk for cord compression, other means of ablating testosterone might be considered, such as ketoconazole, orchiectomy, or LHRH antagonists. Clinical flare responses, as opposed to biochemical flare responses, are very rare during LHRH analog therapy for stage D0 disease and/or in the setting of neoadjuvant hormonal therapy.

Effects of Bloom's syndrome fibroblasts on genetic recombination and mutagenesis of herpes simplex virus type 1.

The effects of Bloom's syndrome (BS) fibroblasts on genetic recombination and the mutation frequency of herpes simplex virus type 1 (HSV-1) was determined by employing two factor crosses of selected temperature-sensitive (ts) mutants. A significant increase in the recombination frequency (RF) was observed in seven of nine crosses when multiple BS fibroblast lines were compared to normal human fibroblasts. The RF of HSV-1 ts mutants increased following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of normal, but not BS fibroblasts, suggesting that BS fibroblasts express higher constitutive levels of genetic recombination activity. HSV-1 ts mutants demonstrated significantly higher reversion frequencies to the nontemperature sensitive (ts+) phenotype following growth in BS rather than normal fibroblasts, indicating that exogenous viral DNA encoding many of the enzymes necessary for its own replication is affected by the mutator phenotype of BS.

Incorporation of 1-beta-D-arabinofuranosylcytosine into DNA and mutagenesis of herpes simplex virus type 1.

To investigate the association between incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into herpes simplex virus type 1 (HSV-1) DNA and mutagenesis at selected genetic loci, we grew virus in the presence or absence of ara-C and compared the mutation frequencies. Both the forward mutation frequency of HSV-1 (KOS) to the tk- phenotype and the reversion frequency of a temperature-sensitive mutant to the non-temperature-sensitive (ts+) phenotype were significantly increased following growth in ara-C. When the same viruses were grown in an inhibitor of the HSV-1 DNA polymerase that is not incorporated into DNA, no significant increase in the frequency of tk- or ts+ particles was observed. Furthermore, HSV-1 araAr, a mutant resistant to ara-C on the basis of reduced incorporation of the analog into viral DNA, did not demonstrate an increase in the number of tk- particles following growth in ara-C. These results suggest that mutagenesis of HSV-1 following growth in ara-C correlates with incorporation of the nucleoside analog into viral DNA.

Spectrum of cis-diamminedichloroplatinum(II)-induced mutations in a shuttle vector propagated in human cells.

The supF gene of the shuttle vector pZ189 was used as a target for the study of mutations induced by cis-diamminedichloroplatinum(II) (cis-DDP). Normal human repair-proficient fibroblasts and cis-DDP repair-deficient xeroderma pigmentosum (XP) cells were used as host cells to study the effect of cis-DDP on the inhibition of shuttle vector replication and mutagenesis. Transfection of cis-DDP-treated pZ189 into normal and XP cell lines resulted in a marked increase in the mutation frequency and a decrease in the replication efficiency of the vector. However, these effects were much greater for the plasmid propagated in XP cells. Atomic absorption spectroscopy showed that six to eight Pt-DNA adducts per plasmid were necessary to inhibit plasmid replication by 50% in normal cells. In contrast, only one to two Pt-DNA adducts were necessary to inhibit replication of the plasmid by 50% in XP cells. Analysis of mutation sites demonstrated that cis-DDP treatment resulted primarily in single and double mutations separated by one base and limited to a few locations within the 85-bp mature tRNA. Propagation of the cis-DDP-treated vector in either normal or XP cells led to predominantly transversion mutations at AGA, AGG, and GAG sites and a cis-DDP-associated deletion of 174 bp. Although mutations occurred at target sites for cis-DDP adduct formation, there was no correlation between sites of mutation and the most frequent sites of adduct formation.