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Biomedical subjects

T M Chen

Publications and source records attributed to T M Chen.

At least 19 recordsLinked to original sources
PubMed1999

Vascularity index as a novel parameter for the in vivo assessment of angiogenesis in patients with cervical carcinoma.

BACKGROUND: The importance of angiogenesis now is well recognized. Conventionally, tumor angiogenesis is assessed by determination of microvessel density (MVD) in the surgical specimen. This study examines tumor angiogenesis using power Doppler ultrasound and a quantitative image processing system. The authors hope to develop an in vivo and noninvasive method for quantitating tumor angiogenesis. METHODS: Thirty-five patients with FIGO Stage IB-IIA cervical carcinoma exhibiting visible cervical tumors by transvaginal ultrasound were included in this study. All patients underwent radical abdominal hysterectomy and pelvic lymph node dissection. Transvaginal power Doppler ultrasound was performed before surgery to search for blood flow signals from the tumor. The intratumoral vascularity index (VI) and resistance index (RI) were calculated. The VI was defined as the number of colored pixels divided by the number of total pixels in the defined tumor section. Maximal VI and minimal RI of a certain tumor were used for analysis. Clinical and pathologic data also were recorded. The MVD of the excised tumor was assessed immunohistochemically using a monoclonal antibody against CD34. RESULTS: Significantly higher VI values were noted in Stage II tumors compared with Stage 1 tumors (19.01+/-10.90% vs. 9.09+/-6.59%; P = 0.008), tumors invad-ing+/-50% of the cervical stroma compared with tumors invading < 50% of the cervical stroma (13.20+/-8.20% vs. 5.72+/-5.00%; P = 0.003), tumors with lymphovascular emboli compared with tumors without lymphovascular emboli (17.28+/-8.26% vs. 6.98 +/- 5.09%; P = 0.001), and tumors with pelvic lymph node metastases compared with tumors without pelvic lymph node metastases (26.16+/-7.88% vs. 8.00+/-4.95%; P = 0.021). None of the variables mentioned earlier showed a significant difference in terms of the RI values. No correlation was noted between intratumoral RI and VI in respective tumors (P = 0.53). Analysis of VI revealed linear regression with regard to tumor size (P < 0.001, correlation coefficient [r] = 0.586) and depth of stromal invasion (P = 0.002, r = 0.497). In addition, the MVD exhibited a linear relation with VI (P = 0.006, r = 0.454), tumor size (P = 0.005, r = 0.465), and depth of stromal invasion (P = 0.009, r = 0.436) using simple regression analysis. No correlation could be found between MVD and RI. CONCLUSIONS: In cervical carcinoma, intratumoral VI assessment by power Doppler ultrasound and quantitative image processing system showed better correlation with tumor stage, tumor size, and pathologic findings including depth of stromal invasion, lymphovascular emboli, and pelvic lymph node metastases than intratumoral RI. The in vivo indicator of angiogenic activity (VI) is well correlated with the conventional indicator of tumor angiogenic activity (MVD). Thus, VI could be a useful parameter for the in vivo assessment of global tumor angiogenesis.

Adenocarcinoma↗
PubMed1999

Sternocleidomastoid pseudotumor and congenital muscular torticollis in infants: a prospective study of 510 cases.

OBJECTIVES: Congenital muscular torticollis is a common and controversial condition in infancy. We studied prospectively a group of infants with clearly defined sternomastoid tumor treated with a well-defined protocol. STUDY DESIGN: A total of 510 cases of sternomastoid tumor in infants over a 10-year period were studied prospectively with a mean follow-up of 3.5 years and a range from 1. 5 years to 13 years. The clinical presentations, associated abnormalities, treatments, and outcomes of the overall group and subgroups were evaluated to determine the most important prognostic factors. RESULTS: The mean age of presentation was day 24, with most (92.7%) presenting before the age of 3 months. There was a high correlation with breech presentation and assisted delivery. Clinical subgroups according to the deficit in passive rotation of the neck correlated with the incidence of hip dysplasia (up to 11.6%), mode of delivery, time of presentation, degree of craniofacial asymmetry, head tilt, and the size and extent of the pseudotumor (P <.05). With an early and prolonged manual stretching program, 90.7% had excellent and good overall results. The 6.7% of patients in the poor outcome group requiring surgical treatment all belonged to the more severe rotation limitation groups. CONCLUSIONS: Subgrouping patients with sternomastoid tumor according to the passive limitation of rotation of the neck has prognostic significance, with good overall results of conservative treatment with manual stretching.

Diagnosis, Differential↗
PubMed1999

The merit of sintered PDLLA/TCP composites in management of bone fracture internal fixation.

Polyesters based on lactic acid have been reported in terms of safety and biodegradation in human beings for 2 decades. The greatest advantage of such material is its degradation conducted only by hydrolysis, whereby the ester backbones are supposed to be unchained in the aqueous condition. The final degradable products are carbon dioxide and water which can be metabolized and digested in the physiological environment. The goal of this study was aimed at developing a composite sintered with poly-DL-lactide (PDLLA) and tricalcium phosphate (TCP) ceramic particles for orthopedic application. The TCP particles in a range of 30-60 wt% with 5 wt% increments were doped into the PDLLA matrix which was prepared by melting and hot pressing techniques for the reinforcement. The basic mechanical strength, biodegradable behavior, and biological response of the composites were investigated in the study. Various techniques such as pH meter, UV, Fourier-transform infrared, and x-ray diffractometer were used to examine and record the degradable process of the composites soaked in saline for 1-16 weeks. The rabbit femur condyle fracture fixation test was used to evaluate tissue compatibility and the effects of bone fracture fixation on the composites. Histological observation and x-ray photography were used for investigating assistance. The mechanical strength of the composites initially increased with TCP additions up to 50wt%, but thereafter they showed no significant difference (p < 0.05). The composite with 50 wt% TCP addition showed greater mechanical strength and had good agreement with cortical bone in terms of its elastic modulus of 30-40 GPa. The weight loss of the pure PDLLA soaked in the saline started at 4 weeks and reached 95% after 16 weeks. The composites compared with pure PDLLA, however, showed no apparent evidence of degradation after soaked for 12 weeks. The possible mechanisms for the delayed degradation of the composites in saline might have been solution penetration retardation by the ceramic particles and chemical bonds formed between the interface of the TCP particles and the PDLLA matrix. In the histological evaluation of the rabbit femur condyle fracture fixation test, the surface of the composite with 50 wt% TCP addition was attached by the newly generated bone without fibrous tissue around 8 weeks after implantation. The fractured bone was gradually healed and the composite firmly and properly fixed on the fracture area during the implanted period, which provided a breeding environment for normal bone remodeling. The developed composite was thought to be an alternative material for orthopedic application in the future, especially for bone screws and bone plates.

Absorption↗
PubMed1999

Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex.

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.

Animals↗
PubMed1998

Clinical application of intratumoral blood flow study in patients with endometrial carcinoma.

BACKGROUND: The objective of this study was to evaluate the correlation between intratumoral blood flow as assessed by color Doppler ultrasound with stage, tumor grade, depth of invasion, and lymph node metastasis in endometrial carcinoma and determine its clinical usefulness. METHODS: Sixty-six patients with endometrial carcinoma were enrolled. All patients received surgical treatment. Transvaginal color Doppler ultrasound was performed before surgery to detect the arterial blood flow signals within the tumors and the lowest resistance index (RI) was recorded. Formalin fixed, paraffin embedded pathology slides were reviewed by a senior pathologist to evaluate the histologic grading, tumor size, depth of myometrial invasion, and presence of lymph node metastasis. RESULTS: Intratumoral RI correlated well with surgical staging, histologic grading, the depth of myometrial invasion, and the presence of lymph node metastasis. Significantly lower RI was noted in tumors of advanced stage (> than International Federation of Obstetrics and Gynecology [FIGO] Stage I) (0.38 +/- 0.09 vs. 0.54 +/- 0.11; P < 0.001), tumors with higher histologic grade (Grade 3) (0.36 +/- 0.08 vs. 0.53 +/- 0.11; P < 0.001), tumors with deep myometrial invasion (> 50% myometrial thickness) (0.38 +/- 0.07 vs. 0.54 +/- 0.11; P < 0.001), and tumors with lymph node metastasis (0.34 +/- 0.07 vs. 0.52 +/- 0.11; P < 0.001) compared with tumors with Stage I, Grade 1/2 histology, no or superficial myometrial invasion, and absence of lymph node metastasis, respectively. CONCLUSIONS: Intratumoral blood flow analysis assessed by color Doppler ultrasound correlates well with surgical stage, tumor grade, myometrial invasion, and lymph node metastasis in patients with endometrial carcinoma. Assessment of tumor angiogenesis using color Doppler ultrasound provides useful information for the preoperative prediction regarding stage, histologic grade, depth of myometrial invasion, and presence of lymph node metastasis in patients with endometrial carcinoma. The authors believe routine pelvic lymph node dissection should be performed for those patients whose lowest RI values of intratumoral blood flow are < or = 0.4 because those patients are at very high risk for pelvic lymph node involvement.

Adult↗
PubMed1998

Induction of stress response and differential expression of 70 kDa stress proteins by sodium fluoride in HeLa and rat brain tumor 9L cells.

We herein demonstrate that sodium fluoride (NaF) acts as a stress response inducer on HeLa and 9L rat brain tumor cells. NaF is only slightly cytotoxic, and inhibitory to Ser/Thr-phosphatases but not to Tyr-phosphatases in both cell lines. After treatment with 5 mM NaF for 2 h, the phosphorylation levels of vimentin and an alkali-resistant 65-kDa phosphoprotein were enhanced, a common phenomenon detected in cells under a variety of stress conditions. Under an identical treatment protocol, in which the cells were treated with 5 mM NaF for 2 h and then allowed to recover under normal growing conditions for up to 12 h, NaF differentially induced the cytoplasmic/nuclear heat-shock protein70s (including both the inducible and the constitutively expressed members of this protein family) in HeLa cells and the endoplasmic reticulum residing heat-shock protein70 (the glucose-regulated protein with an apparent molecular weight of 78 kDa) in 9L cells. Electrophoretic mobility shift assays (EMSA) using probes containing well-characterized regulatory elements revealed the activation of the heat-shock factor in HeLa but not in 9L cells; this is in good agreement with the stress protein induction pattern. Additional differential induction of binding activities toward EMSA probes individually containing NF-KB, AP-2, and CRE-like elements were detected in NaF-treated cells. The possible involvement of these binding sites as well as the corresponding factors in the stress response are discussed.

Animals↗
PubMed1998

Preoperative ultrasound study in predicting lymph node metastasis for endometrial cancer patients.

OBJECTIVE: The objective of this study was to evaluate the efficacy of preoperative ultrasound (US) findings such as tumor size, status of myometrial invasion, and intratumoral "resistance index" (RI) in predicting lymph node metastasis in endometrial carcinoma patients. METHODS: Forty-two patients with endometrial cancer were enrolled. All patients underwent total abdominal hysterectomy, pelvic lymph node dissection or sampling, and para-aortic lymph node sampling. Two-dimensional and color Doppler US were performed before surgery to measure tumor size, depth of myometrial invasion, and intratumoral arterial RI. Formalin-fixed, paraffin-embedded pathologic slides from surgical specimens were reviewed by a senior pathologist to evaluate histologic type and grade, depth of myometrial invasion, cervical involvement, lymph-vascular emboli, and status of lymph node metastasis. RESULTS: There were 12 patients with pelvic and/or para-aortic lymph node metastases and 30 patients without nodal metastases. Patients with tumors larger than 2.5 cm by US (11/12 vs 14/30, P = 0.008), more than half myometrial invasion by US (9/12 vs 5/30, P < 0.001), and intratumoral RI values less than 0.4 by US (12/12 vs 4/30, P < 0.001) had a significantly higher incidence of nodal metastases as compared with patients with tumors smaller than 2.5 cm, no or superficial myometrial invasion, and RI values higher than 0.4, respectively. Multiple regression analysis showed that only intratumoral RI values less than 0.4 were significantly correlated with nodal metastasis (P < 0.001, r2 = 0. 650). We used the intratumoral RI value as the parameter to evaluate nodal metastasis in endometrial cancer patients. Twelve of sixteen patients with intratumoral RI values <0.4 had a high incidence of nodal metastases. None of the 26 patients with intratumoral RI values >0.4 had nodal metastases. CONCLUSIONS: Preoperative ultrasound features can offer important information for predicting lymph node metastasis in endometrial cancer patients. Patients with tumors with intratumoral RI values less than 0.4 should be highly suspected of having lymph node metastases and further management such as pelvic lymph node dissection or postoperative pelvic radiotherapy would be needed for these patients.

Adult↗
PubMed1998

Skin injury in the operating room.

Skin injury occurring in the operating room may occur as a result of electrical current, thermal injury, chemical irritation and mechanical stress. Between 1 December 1996 and 28 February 1997, 19 cases of skin injury from a total of 3657 operations were noted in our hospital. These injuries tend to be ignored because of their early spontaneous healing. We suggest that medical staff should pay more attention to this complication and prevent it from occurring.

Adult↗
PubMed1998

The comparison of early fluid therapy in extensive flame burns between inhalation and noninhalation injuries.

Over the last half century, advances in treatment have changed the principal cause of death in burn patients from burn shock and wound sepsis to pulmonary sepsis, of which inhalation injury has always played a key role in morbidity and mortality. Even though Navar et al., Am. J. Surg. 1985;150:716-720 have noted that patients with inhalation injury had a mean fluid requirement of 5.8 ml/kg/% burn to achieve resuscitation from early burn shock, while patients without inhalation injury required only a mean fluid of 4.0 ml/kg/% burn, to achieve successful resuscitation in inhalation injured patients with minimum but adequate fluid has always been a challenge. To further define the difference of early fluid therapy between inhalation and noninhalation in extensively burned patients, a retrospective analysis was carried out in the Tri-Service General Hospital. Sixty-two flame burned patients (aged from 16 to 81 years-old with a mean age of 33.2+/-15.1 years: with burn size ranging from 30% to 98% TBSA with a mean burn size of 60.5+/-22.7%; 26 with inhalation injury; noninhalation 36) were reviewed during a 5-year period. The Parkland formula is the initial choice of fluid regimen with 4 ml/kg/% burn and the amount of replacement is monitored by urine output and is titrated to maintain urine output between 0.5 and 1.0 ml/kg/h. The mean amounts of fluid requirements of both inhalation and noninhalation burned patients were 3.1 +/- 1.0 and 2.3+/-0.8 ml/kg/% burn respectively (p < 0.05). Our study showed less fluid requirement for both inhalation and noninhalation injured patients in comparison with the Navar study and Parkland predictions in the first 24 h postburn. Furthermore, the inhalation injured patients definitely required volumes of fluid in excess of those required in noninhalation injured cases.

Adolescent↗
PubMed1998

Concomitant alterations in distribution of 70 kDa heat shock proteins, cytoskeleton and organelles in heat shocked 9L cells.

Maintenance of cell architecture and positioning of organelles are major functions of the cytoskeleton. On the other hand, induction of heat shock proteins (HSPs) and reorganization of the cytoskeleton are the most significant changes in heat-shocked mammalian cells. We examine the alterations in HSP70 and its constitutively expressed cognate, HSC70, as well as the cytoskeleton and organelles in 9L rat brain tumor cells upon heat shock. We employed fluorescence microscopy and scanning electron microscopy to follow these changes. Levels of HSP70s were quantified by Western blotting. Accumulation of HSC70 was more transient and the protein translocated to and subsequently exited from the nucleus more rapidly than HSP70. Changes in actin microfilaments include the nuclear localization of actin fraction and disappearance of cytoplasmic microfilament bundles, while the cortical actin microfilaments were almost unaffected. Furthermore, microtubules retracted slightly from the cell periphery but remained largely unchanged. In contrast, the intermediate filaments collapsed into the perinuclear region. The mitochondria converted from filamentous into granular forms and clustered in a region overlapping with the collapsed intermediate filaments. All of the above alterations are reversible and largely reverted after 8 h of recovery. The effect on Golgi organization was very transient and the apparatus assumed a normal appearance within 4 h after the heat treatment. The ER, on the other hand, was totally unaffected by the heat treatment. These observations help correlate the sequential events following a stress like heat shock and suggest possible physiological functions of these essential constituents of a cell under stress.

Animals↗
PubMed1998

Benzylacyclouridine enhances 5-fluorouracil cytotoxicity against human prostate cancer cell lines.

At a nontoxic growth inhibitory concentration benzyloxyacyclouridine (BAU), a potent and specific inhibitor of uridine phosphorylase (UrdPase), enhanced 5-fluorouracil (5-FU) cytotoxic activity against human prostate cancer PC-3 and DU-145 cell lines. The BAU/5-FU combination exhibited greater antitumor activity in vivo using PC-3 human xenografts compared to 5-FU alone, with no associated increase in animal host toxicity. The mechanism(s) responsible for the enhanced in vitro and in vivo activity of this combination may involve enhanced formation of the 5-FU nucleotide metabolites FdUMP, FdUTP, and FUTP resulting in enhanced inhibition of thymidylate synthase (TS) and increased incorporation of fluoropyrimidine metabolites into tumoral RNA and DNA.

Animals↗
PubMed1998

Predictive sensitivity of human cancer cells in vivo using semipermeable polysulfone fibers.

An in vivo experimental model was developed to predict efficiently and accurately chemosensitivity of human tumors. Human cancer cells either from cultured cell lines or from patients' tumors were injected directly into semipermeable polysulfone fibers subsequently implanted into immunocompetent rats. Results suggest utility of this novel model system for predicting tumor sensitivity to a wide range of anticancer agents and for potentially guiding the treatment of cancer patients in the clinical setting.

Animals↗
PubMed1997

Intrathecally administered c-fos antisense oligodeoxynucleotide decreases formalin-induced nociceptive behavior in adult rats.

c-fos antisense strategy was applied as a pharmacological approach to characterize its dose-dependent role and reversibility in the reduction of formalin-induced hyperalgesia. Nociceptive behavioral responses (weighted score, flinching response, licking/biting) following formalin (50 microl 5%) injection were assessed in adult Wistar rats receiving different doses (50 nM, 250 nM) of intrathecally administered c-fos antisense oligodeoxynucleotides at different times prior to formalin injections. The treatments dose dependently decreased both Fos immunoreactivity expression in dorsal horn of rat lumbar spinal cord and all nociceptive measures in the tonic phase of the formalin test. c-Fos correlated well with weighted pain score and/or flinching responses, but not with licking/biting behavior. With the exception of a 48-120 h period required for licking/biting behavior to be restored to its normal status, the suppressive effect on c-fos expression and other nociceptive behaviors disappeared 48 h following c-fos antisense oligodeoxynucleotide treatment. The results suggest a pharmacological potential of c-fos antisense oligodeoxynucleotides in the central nervous system to block immediate-early genes and their resulting physiological consequence following noxious stimulus.

Animals↗
PubMed1997

Development and prospective validation of a clinical index to predict survival in ambulatory patients referred for cardiac transplant evaluation.

BACKGROUND: Risk stratification of patients with end-stage congestive heart failure is a critical component of the transplant candidate selection process. Accurate identification of individuals most likely to survive without a transplant would facilitate more efficient use of scarce donor organs. METHODS AND RESULTS: Multivariable proportional hazards survival models were developed with the use of data on 80 clinical characteristics from 268 ambulatory patients with advanced heart failure (derivation sample). Invasive and noninvasive models (with and without catheterization-derived data) were constructed. A prognostic score was determined for each patient from each model. Stratum-specific likelihood ratios were used to develop three prognostic-score risk groups. The models were prospectively validated on 199 similar patients (validation sample) by calculation of the area under the receiver operating characteristic curve for 1-year event-free survival, the censored c-index for event-free survival, and comparison of event-free survival curves for prognostic-score risk strata. Outcome events were defined as urgent transplant or death without transplant. The noninvasive model performed well in both samples, and increased performance was not attained by the addition of catheterization-derived variables. Prognostic-score risk groups derived from the noninvasive model in the derivation sample effectively stratified the risk of an outcome event in both samples (1-year event-free survival for derivation and validation samples, respectively: low risk, 93% and 88%; medium risk, 72% and 60%; high risk, 43% and 35%). CONCLUSIONS: Selection of candidates for cardiac transplantation may be improved by use of this noninvasive risk-stratification model.

Cardiac Output, Low↗
PubMed1997

Protein synthesis inhibitor cycloheximide dose-dependently decreases formalin-induced c-Fos protein and behavioral hyperalgesia in rats.

We had previously demonstrated that c-fos antisense oligodeoxynucleotides dose-dependently suppressed formalin-induced c-Fos protein and behavioral hyperalgesia. To test whether de novo protein synthesis is required for the development of persistent pain after peripheral inflammation, we observed formalin-induced spinal c-Fos protein and nociceptive behaviors following pretreatment with cycloheximide, a protein synthesis inhibitor. Cycloheximide dose-dependently inhibited formalin-induced spinal c-Fos protein and tonic nociceptive responses. The possible non-specific effects other than protein synthesis inhibition on nociceptive behavior were carefully discussed and excluded. These results provide further support to the hypothesis that de novo protein synthesis is essential for the development of behavioral hyperalgesia.

Animals↗
PubMed1997

Recipient bed vascularity and the survival of ischaemic flaps.

The purpose of this study was to identify the length of the ischaemic period required to induce the 'no-reflow' phenomenon in a rat epigastric flap on an avascular recipient site. The vascularity of the recipient bed may affect flap survival in the early postischaemic stage after flap transfer. Initially, we designed epigastric flaps in 300-350 g Sprague-Dawley rats and separated the rats into four groups of 5 rats each (total 20 rats). In groups 1, 2, 3, the flaps were made ischaemic for 1 hour, 6 hours and 10 hours, respectively, by temporarily clipping the epigastric artery and vein. In group 4, the epigastric artery and vein were divided to create permanent ischaemia. In groups 1, 2 and 3, ischaemia was ended by removing the clips. After the ischaemic flaps were reperfused, their viability was studied by measuring the flap survival rate at postoperative day 7. Flap survival was studied by direct observations, laser Doppler flowmeter measurement of flap blood flow, histopathology, and carbon particle perfusion of the flap vasculature. Ischaemic flaps of groups 1 and 2 recovered almost completely after reperfusion due to the short period of ischaema. In a second series of experiments, in order to evaluate the contribution to flap survival of the recipient vascularised bed, another four groups of epigastric flaps (of 5 animals each, using the same time periods as above) were raised and a piece of Biobrane was interposed between the flap and the recipient bed before the flap wound was closed, to eliminate all nutrient supply from the recipient bed. THe results showed that the combined effect of the reperfused flap vasculature plus the metabolic contribution of the recipient bed significantly (P < 0.01) increased the extent of flap survival of the 6- and 10-hour ischaemic flaps as well as the divided pedicle flaps, which were never reperfused. An absolute 'no-reflow' rat model flap for further flap salvage studies was also developed.

Animals↗
PubMed1997

Comparative analyses of the latency-associated transcript promoters from herpes simplex virus type 1 strains H129, +GC and KOS-63.

We have analyzed the activity of a specific portion of the latency-associated transcript (LAT) promoter of three strains of herpes simplex virus type 1 (HSV-1) in neuronal and non-neuronal cell types. Restriction fragments containing the LAT promoter sequences and the 5'-end of the LATs were isolated from HSV-1 strains H129, +GC and KOS-63, sequenced and cloned into a chloramphenicol transferase (CAT) plasmid vector. These vectors were separately assayed for CAT production in human (SknSH) and mouse (C-1300) neuroblastoma cell lines and a human continuous cell line (HeLa). Strain KOS-63 contained a C to T base substitution within the LAT promoter binding factor element upstream of the cAMP response element binding sequence. In replicate experiments, in which the construct DNA was used for transfection, the CAT constructs from strains H129 and +GC functioned equally well in all three cell lines. In contrast, the strain KOS-63 CAT construct functioned significantly better in HeLa cells than in neuroblastoma cell lines and better than the identical CAT constructs from strains H129 and +GC. In addition, the construct from strain KOS-63 functioned less well in the human neuroblastoma cell line than in HeLa or C-1300 neuroblastoma cells. When LAT expression was examined directly in vivo by in situ hybridization, strain KOS-63 produced slightly less LAT RNA than strain H129 within trigeminal ganglionic neurons of latently infected rabbits. However, utilizing competitive gel-shift assays, DNA fragments containing the LAT promoter binding element from all three strains bound equivalent amounts of HeLa cell nuclear proteins. Together, these results suggest that the activity expressed by the strain KOS-63 LAT promoter in vivo and in vitro may relate to positive or negative effects of DNA binding proteins on LAT transcription, and that these effects are cell-type dependent.

Animals↗