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Biomedical subjects

R M Hoffman

Publications and source records attributed to R M Hoffman.

At least 19 recordsLinked to original sources
PubMed2000

Methioninase gene therapy of human cancer cells is synergistic with recombinant methioninase treatment.

Results obtained over the past 40 years have demonstrated that tumor cells of all types tested have an elevated growth requirement for methioninase compared with normal cells. Recombinant methioninase (rMETase) cloned from Pseudomonas putida has been found previously to be an effective antitumor agent attributable to deprivation of the extracellular methionine source of the tumor. To degrade intracellular methioninase, we have now developed an adenoviral vector inserted with the P. putida methioninase (MET) gene (rAd-MET). The in vitro efficacy of rAd-MET was tested on the OVCAR-8 human ovarian cancer cell line, the HT1080 human fibrosarcoma cell line, and human normal fibroblasts. rAd-MET transduction of OVACAR-8 and HT1080 resulted in high levels of methioninase expression up to 10% or more of the total protein of the cells, depending on the multiplicity of infection. The IC50 of rAd-MET for OVCAR-8 cells in 96-well plates was approximately 2 x 106 plaque-forming units (pfu)/well. The IC50 of control adenovirus (control-rAd) was 4 x 10(7) pfu/well, 20 times higher than rAd-MET. In the presence of the IC50 of 2 x 10(6) pfu/well of rAd-MET, the addition of 0.025 units/ml of rMETase, which is 25% of the IC50, resulted in a 90% inhibition of tumor cell number. This indicated that rAd-MET enhanced the efficacy of rMETase. In contrast, 2 x 10(6) pfu/well of control-rAd in combination with 0.025 units/ml of rMETase had an efficacy of only 10% inhibition of cell number. The synergistic effect of the combination of rMETase and rAd-MET was quantitated by calculating the combination index (CI). The CIs for all combinations of rAd-MET and rMETase tested on OVCAR-8 were <0.7 with a mean of 0.5, indicating synergy. Similar synergy of rAd-MET and rMETase was seen on HT1080 human fibrosarcoma cells with a mean of 0.74. In contrast, the CIs of all combinations of rMETase and control adenovirus concentrations tested on both cell lines had a mean CI of approximately 1, which indicated that this combination had only an additive effect. The normal fibroblasts, on the other hand, appeared relatively resistant to the MET gene because in the presence of rMETase, 2.5 x 10(7) pfu/well of rAd-MET or control rAd had almost an identical effect on cell survival. The selectively strong synergy of rAd-MET and rMETase on cancer cells allows reduced levels of each agent to be used, thus decreasing potential side effects.

Carbon-Sulfur Lyases↗
PubMed2000

Whole-body optical imaging of green fluorescent protein-expressing tumors and metastases.

We have imaged, in real time, fluorescent tumors growing and metastasizing in live mice. The whole-body optical imaging system is external and noninvasive. It affords unprecedented continuous visual monitoring of malignant growth and spread within intact animals. We have established new human and rodent tumors that stably express very high levels of the Aequorea victoria green fluorescent protein (GFP) and transplanted these to appropriate animals. B16F0-GFP mouse melanoma cells were injected into the tail vein or portal vein of 6-week-old C57BL/6 and nude mice. Whole-body optical images showed metastatic lesions in the brain, liver, and bone of B16F0-GFP that were used for real time, quantitative measurement of tumor growth in each of these organs. The AC3488-GFP human colon cancer was surgically implanted orthotopically into nude mice. Whole-body optical images showed, in real time, growth of the primary colon tumor and its metastatic lesions in the liver and skeleton. Imaging was with either a trans-illuminated epifluorescence microscope or a fluorescence light box and thermoelectrically cooled color charge-coupled device camera. The depth to which metastasis and micrometastasis could be imaged depended on their size. A 60-microm diameter tumor was detectable at a depth of 0.5 mm whereas a 1, 800-microm tumor could be visualized at 2.2-mm depth. The simple, noninvasive, and highly selective imaging of growing tumors, made possible by strong GFP fluorescence, enables the detailed imaging of tumor growth and metastasis formation. This should facilitate studies of modulators of cancer growth including inhibition by potential chemotherapeutic agents.

Adenocarcinoma↗
PubMed2000

A novel approach to gene therapy of albino hair in histoculture with a retroviral streptomyces tyrosinase gene.

In order to induce melanin production in mammalian cells with pigment disorders such as albino hair, a recombinant retrovirus containing the mel locus of Streptomyces antibioticus was constructed. The S. antibioticus mel locus, which consists of the open reading frame (ORF)-438 and the tyrosinase gene, was specifically derived by polymerase chain reaction (PCR) from Streptomyces plasmid pIJ702. The ORF-438 is required for the transfer of copper to apotyrosinase, which is essential for tyrosinase enzymatic activity. The tyrosinase gene was inserted into the XhoI/BamHI cloning site of the pLXSN retroviral vector to obtain pLtyrSN. An internal ribosome entry site (IRES) suitable for mammalian cell expression was obtained from the pLXIN retroviral vector by PCR. The ORF-438 and IRES DNA fragments were inserted into the pLtyrSN vector to obtain the tyrosinase-expression retroviral vector pLmelSN. The expression vector was amplified in murine PT67 packaging cells, where the ORF-438 and tyrosinase genes were also co-expressed as determined by reverse transcription-PCR. In order to evaluate the vector's ability to restore pigment production in cells with a pigment disorder, albino-mouse skins were histocultured and then infected with the pLmelSN retrovirus. Six days after infection, melanin granules were observed in approximately 60% of albino-mouse hair follicles in the histocultured skin. These results demonstrated that the S. antibioticus mel operon could express an active tyrosinase and produce melanin in the albino-mouse hair follicles. This novel gene therapy approach, using a small and simple tyrosinase operon in a high-expression vector, has a potentially wide application for therapy of pigment disorders in hair follicles.

Albinism↗
PubMed2000

Survival efficacy of the combination of the methioninase gene and methioninase in a lung cancer orthotopic model.

We have previously demonstrated the antitumor efficacy of recombinant methioninase (rMETase) derived from Pseudomonas putida. To enhance the efficacy of rMETase, we have constructed the pLGFP-METSN retrovirus encoding the P. putida methioninase (MET) gene fused with the green fluorescent protein (GFP) gene. pLGFP-METSN or control vector pLGFPSN was introduced into the human lung cancer cell line H460. The methionine level of H460-GFP-MET cells was reduced to 33% of that of H460-GFP cells. rMETase (0.08 U/mL) in the medium resulted in 10% survival of H460-GFP-MET cells compared with untreated cells in vitro. In contrast, rMETase-treated H460-GFP cells survived at 90% of control. Tissue fragments harvested from subcutaneous tumors of H460-GFP-MET or H460-MET were implanted by surgical orthotopic implantation into the lungs of nude mice. A suboptimal dose of rMETase was administered intraperitoneally daily to mice in each group. Overall survival of rMETase-treated animals with H460-GFP-MET tumors was significantly longer than either rMETase-treated or -untreated animals with H460-GFP tumors (P < .05 in log-rank test). In two repeat experiments, rMETase-treated animals with H460-GFP-MET tumors had a 30-day survival of 80% and 83%, respectively. Untreated animals with H460-GFP-MET tumors had a 30-day survival of 40% and 58%, respectively. rMETase-treated animals with H460-GFP tumors had a 30-day survival of 0% and 33%, respectively. Untreated animals with H460-GFP tumors had a 30-day survival of 0% and 33%, respectively. The retrovirus-mediated gene transfer of METase decreased the intracellular methionine level of tumor cells and consequently enhanced the efficacy of treatment with the rMETase protein. We have thus demonstrated a new strategy of combination tumor therapy with the gene and gene product of MET.

Animals↗
PubMed2000

How common are various causes of dizziness? A critical review.

BACKGROUND: Although dizziness is a common symptom in both primary care and referral practices, the relative frequency of various causes has not been well delineated. METHODS: A MEDLINE search identified 12 articles containing original data on the etiology of dizziness in consecutive patients. Study sites included primary care offices (n = 2), emergency room (n = 4), and referral clinics (n = 6). Each study's strength of design was graded using nine quality criteria. RESULTS: Dizziness was attributed to a peripheral vestibulopathy in 44% of patients, a central vestibulopathy in 11%, psychiatric causes in 16%, other conditions in 26%, and an unknown cause in 13%. Certain serious causes were relatively uncommon, including cerebrovascular disease (6%), cardiac arrhythmia (1.5%), and brain tumor (<1%). CONCLUSIONS: Dizziness is due to vestibular or psychiatric causes in more than 70% of cases. Since serious treatable causes appear uncommon, diagnostic testing can probably be reserved for a small subset of patients.

Adult↗
PubMed2000

Antimetastatic intraoperative chemotherapy of human colon tumors in the livers of nude mice.

We have developed a new antimetastatic chemotherapeutic strategy for combination with hepatic resection of human colon cancers in a high-metastasis nude mouse model. The new procedure involves i.p. administration of 5-fluorouracil (5-FU) 2 h before hepatic resection of the human colon tumors, with therapy continued postoperatively for 4 consecutive days. We termed this strategy neo-neoadjuvant chemotherapy. The regime significantly prolonged animal survival compared with preoperative 5-FU neoadjuvant therapy, 5-FU postoperative adjuvant therapy, surgery alone, 5-FU without surgery, or the untreated control. The median survival of neo-neoadjuvant i.p. 5-FU-treated group was 81 days, compared with 27 days for the control group (P < 0.009). The median survival of animals in the neoadjuvant group was 37 days (P < 0.021 compared with the control group). There was also a significant difference between the median survival of neo-neoadjuvant, and the neoadjuvant group (P < 0.031). When all animals in the control group had died, 70% of animals with neo-neoadjuvant and 60% of animals with neoadjuvant 5-FU were still alive (P < 0.003 and P < 0.011, respectively). When all animals with neoadjuvant 5-FU treatment had died, 70% of animals with neo-neoadjuvant treatment were still alive (P < 0.003). Survival of all other treatment groups, including 5-FU without surgery, surgery alone, and adjuvant postoperative chemotherapy, was not significantly different from the untreated control group. Two animals in the neo-neoadjuvant group were free of tumors when sacrificed at days 154 and 165 post surgery. Whereas 100% of animals in the control, 90% in the 5-FU alone, 70% in the surgery alone, 60% in the 5-FU adjuvant, and 40% in the neoadjuvant groups had metastases in the lymph nodes draining the liver, only 10% of animals in the neo-neoadjuvant group had metastases. These data suggest that the neo-neoadjuvant therapy increased survival by preventing metastasis of cancer cells not removed in the liver resection procedure. The results of this study indicate that the neo-neoadjuvant treatment strategy for resection of colon cancer liver metastasis should be explored clinically.

Animals↗
PubMed2000

An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.

Whether liver metastases from colon cancer are capable of metastasizing to other sites is an important question in surgical oncology. To answer this question, we have developed a highly metastatic orthotopic transplant model of a liver metastasis from a human colon cancer patient in nude mice that targets the liver and lymph nodes. The metastatic human tumor was transplanted in athymic nude mice by surgical orthotopic implantation (SOI) of a liver metastasis from a colon cancer patient. The human colon tumor was then subsequently implanted in the colon by SOI or, in an additional series of nude mice, in the liver by surgical hepatic implantation (SHI). The mice were then explored over time for lymph node involvement beginning 10 days after implantation. After SOI, 100% of the animals had liver metastasis within 10 days, and subsequently, 19 days after SOI, all lymph nodes draining the liver were involved with metastasis without any retroperitoneal or lung tissue involvement. After SHI, all sites of lymphatic drainage of the liver, including portal, celiac, and mediastinal lymph nodes, were massively involved by metastasis in 100% of the animals as early as 10 days after tumor implantation on the liver. The results of this study demonstrate that liver metastases from colon cancer are capable of remetastasizing to other sites. This study thus suggests that in colon cancer patients with liver metastasis, mediastinal, celiac, and portal lymph node metastases originate from the liver metastasis and not, as previously thought, from primary colon cancer.

Adenocarcinoma↗
PubMed1999

High-malignancy orthotopic nude mouse model of human prostate cancer LNCaP.

BACKGROUND: An animal model of human prostate cancer LNCaP demonstrating high rates of spontaneous metastasis from the orthotopic site after tumor implantation would be very valuable for mechanistic and drug discovery studies. We previously developed microsurgical techniques to implant histologically intact tumor tissues orthotopically in nude mice in order to develop high metastatic mouse models of human cancer. METHODS: Intact tissue of the androgen-dependent human prostate cancer cell line, LNCaP, was implanted on the ventral lateral lobes of the prostate gland by surgical orthotopic implantation (SOI) in a series of 20 nude mice. Mice were autopsied, and histopathological examination of primary tumors and relevant organs was done to identify and quantitate micrometastasis. RESULTS: Eighteen of 20 animals transplanted with LNCaP by SOI had tumor growth. Mean primary tumor weight in the prostate was 9.24 g at time of necropsy. Sixty-one percent of the transplanted animals had lymph node metastasis. Forty-four percent had lung metastasis. Mean survival time was 72 days, indicating a high degree of malignancy of the tumor. CONCLUSIONS: The extensive and widespread lung metastasis as well as lymph node metastasis following orthotopic implantation of LNCaP in nude mice and the short survival time provide a high-malignancy nude model of the LNCaP human prostate tumor.

Animals↗
PubMed1999

A fluorescent orthotopic bone metastasis model of human prostate cancer.

Here, we report a fluorescent spontaneous bone metastatic model of human prostate cancer developed by surgical orthotopic implantation of green fluorescent protein (GFP)-expressing prostate cancer tissue. Human prostate cancer PC-3 cells were transduced with the pLEIN expression retroviral vector containing the enhanced GFP and neomycin resistance genes. Stable GFP high-expression PC-3 clones were selected in vitro with G418, which were then combined and injected s.c. in nude mice. For metastasis studies, fragments of a single highly fluorescent s.c. growing tumor were implanted by surgical orthotopic implantation in the prostate of a series of nude mice. Subsequent micrometastases and metastases were visualized by GFP fluorescence throughout the skeleton, including the skull, rib, pelvis, femur, and tibia The central nervous system, including the brain and spinal cord, was also involved with tumor, as visualized by GFP fluorescence. Systemic organs, including the lung, plural membrane, liver, kidney, and adrenal gland, also had fluorescent metastases. The metastasis pattern in this model reflects the bone and other metastatic sites of human prostate cancer. Thus, this model should be very useful for the study and development of treatment for metastatic androgen-independent prostate cancer.

Animals↗
PubMed1999

Alpha-tocopherol supplementation for men with existing coronary artery disease: a feasibility study.

BACKGROUND: Recent studies have suggested that alpha-tocopherol supplementation can help reduce the incidence of coronary disease. Our objectives were to determine the feasibility of providing alpha-tocopherol supplements to male veterans with existing coronary artery disease and determine its effects on alpha-tocopherol levels and the susceptibility of low-density lipoprotein (LDL) to oxidation. METHODS: Fifty-seven percent of 138 coronary disease patients were willing to participate in a placebo-controlled trial -25% were already taking antioxidants. Thirty-nine men were randomly assigned to either 400 mg/day of alpha-tocopherol (n = 27) or placebo (n = 12). alpha-Tocopherol levels and LDL oxidation (measured by formation of thiobarbituric acid-reactive substance) were measured at baseline and at 6 months. RESULTS: Thirty-three subjects (22 alpha-tocopherol, 11 placebo) completed the study; 3 subjects withdrew after suffering coronary disease events. Supplement compliance exceeded 90% and alpha-tocopherol was well tolerated. The alpha-tocopherol group had a significantly greater mean increase in lipid-adjusted alpha-tocopherol levels (73% vs. -4.6%, P < 0.0001), but oxidized LDL did not change significantly. CONCLUSIONS: A secondary prevention trial among veterans would be feasible because the rates of enrollment, completion, compliance, and clinical events were high. alpha-Tocopherol supplements did not decrease the susceptibility of LDL to oxidation, suggesting that higher dosages or longer duration of supplementation may be required for secondary prevention.

Aged↗
PubMed1999

Evaluating dizziness.

PURPOSE: To conduct a structured literature synthesis on the etiology, prognosis, and diagnostic evaluation of dizziness, and to suggest a primary-care approach to evaluating this symptom. METHODS: Studies were identified from MEDLINE searches (1966 through 1996) and a manual search of bibliographies from retrieved articles. Two investigators independently abstracted study data. RESULTS: The most common etiologies for dizziness were peripheral vestibulopathies (35% to 55% of patients) and psychiatric disorders (10% to 25% of patients). Cerebrovascular disease (5%) and brain tumors (<1%) were infrequent. The history and physical examination led to a diagnosis in about 75% of patients. At least 10% of patients eluded diagnosis. Symptoms were usually self-limited and not associated with an increased risk of mortality. The diagnostic testing literature, which was often methodologically flawed, suggested that routine laboratory tests as well as cardiovascular and neurologic testing had a low yield in unselected patients. We could not derive evidence-based guidelines for using specialized vestibular function tests such as electronystagmography. CONCLUSIONS: Dizziness is usually a benign, self-limited complaint. When a diagnosis can be made, a careful history and physical examination will usually identify the probable cause. Cardiovascular, neurologic, and laboratory testing should be guided by the clinical evaluation. Rigorous studies are needed to determine the accuracy and utility of specialized vestibular testing.

Audiometry↗