Use of tumor dynamics to clarify the observed variability among biochemical recurrence nomograms for prostate cancer.

BACKGROUND: Nomograms for biochemical recurrence (BCR) of prostate cancer (PC) after radical prostatectomy can yield very different prognoses for individual patients. Since the nomograms are optimized on different cohorts, the variations may be due to differences in patient risk-factor distributions. In addition, the nomograms assign different relative scores to the same PC risk factors and rarely stratify for tumor growth rate. METHODS: We compared BCR-free probabilities from the GPSM model with a cell kinetics (CK) model that uses the individual's tumor state and growth rate. We first created a cohort of 143 patients that reproduced the GPSM patient distribution in Gleason score, Prostate specific antigen (PSA), Seminal vesicle involvement and Margin status since they form the GPSM score. We then performed 143 CK calculations to determine BCR-free probabilities for comparison with the GPSM results for all scores and with four other prominent nomograms for a high-risk patient. RESULTS: The BCR-free probabilities from the CK model agree within 10% with those from the GPSM study for all scores once the CK model parameters are stratified in terms of the GPSM risk factors and the PSA doubling time (PSADT). However, the probabilities from widely used nomograms vary significantly. CONCLUSIONS: The CK model reproduces the observed GPSM BCR-free probabilities with a broad stratification of model parameters for PC risk factors and can thus be used to describe PC progression for individual patients. The analysis suggests that nomograms should stratify for PSADT to be predictive.

Secular trends in hip fracture incidence and recurrence.

UNLABELLED: The decline in hip fracture incidence is now accompanied by a further reduction in the likelihood of a recurrent hip fracture among survivors of the first fracture. INTRODUCTION: Hip fracture incidence is declining in North America, but trends in hip fracture recurrence have not been described. METHODS: All hip fracture events among Olmsted County, Minnesota residents in 1980-2006 were identified. Secular trends were assessed using Poisson regression, and predictors of recurrence were evaluated with Andersen-Gill time-to-fracture regression models. RESULTS: Altogether, 2,752 hip fractures (median age, 83 years; 76% female) were observed, including 311 recurrences. Between 1980 and 2006, the incidence of a first-ever hip fracture declined by 1.37%/year for women (p < 0.001) and 0.06%/year for men (p = 0.917). Among 2,434 residents with a first-ever hip fracture, the cumulative incidence of a second hip fracture after 10 years was 11% in women and 6% in men with death treated as a competing risk. Age and calendar year of fracture were independently associated with hip fracture recurrence. Accounting for the reduction in first-ever hip fracture rates over time, hip fracture recurrence appeared to decline after 1997. CONCLUSION: A recent reduction in hip fracture recurrence is somewhat greater than expected from the declining incidence of hip fractures generally. Additional research is needed to determine the extent to which this can be attributed to improved patient management.

Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur.

We have investigated the ability of exogenous transforming growth factor-beta (TGF-beta) to induce osteogenesis and chondrogenesis, critical events in both bone formation and fracture healing. Daily injections of TGF-beta 1 or 2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis. After cessation of the injections, endochondral ossification occurred, resulting in replacement of cartilage with bone. Gene expression of type II collagen and immunolocalization of types I and II collagen were detected within the TGF-beta-induced cartilage and bone. Moreover, injection of TGF-beta 2 stimulated synthesis of TGF-beta 1 in chondrocytes and osteoblasts within the newly induced bone and cartilage, suggesting positive autoregulation of TGF-beta. TGF-beta 2 was more active in vivo than TGF-beta 1, stimulating formation of a mass that was on the average 375% larger at a comparable dose (p less than 0.001). With either TGF-beta isoform, the dose of the growth factor determined which type of tissue formed, so that the ratio of cartilage formation to intramembranous bone formation decreased as the dose was lowered. For TGF-beta 1, reducing the daily dose from 200 to 20 ng decreased the cartilage/intramembranous bone formation ratio from 3.57 to zero (p less than 0.001). With TGF-beta 2, the same dose change decreased the ratio from 3.71 to 0.28 (p less than 0.001). These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGF-beta to proliferate and differentiate, as occurs in embryologic bone formation and early fracture healing.

Genetic expression of extracellular matrix proteins correlates with histologic changes during fracture repair.

We characterized gene expression in the reparative callus that formed after fracture of the rat femur. The callus was divided into regions of bone formation (hard callus) and cartilage formation (soft callus), and gene expression was examined separately in each region. Expression of extracellular matrix protein genes varied with the progression of repair and differed between hard and soft calluses. Messenger ribonucleic acids (mRNAs) for osteonectin, alkaline phosphatase, and type I procollagen were detected in the hard callus at maximal levels during endochondral ossification and bone remodeling (day 15) and at 50% maximal levels during intramembranous bone formation (day 7). Messenger RNAs for these proteins in the soft callus were detected at low levels during chondrogenesis (day 9) but increased to 80% of maximal levels with chondrocyte hypertrophy and mineralization of the cartilage matrix (day 13). Messenger RNAs for type II procollagen and proteoglycan core protein were detected at maximal levels in the soft callus during chondrogenesis (day 9). Osteocalcin gene expression was detected in the hard callus during endochondral ossification and remodeling but not during intramembranous bone formation or at any time in the soft callus. Osteonectin mRNA was detected in both the hard and soft callus throughout the entire course of fracture repair. Expression of cartilage and bone-related genes correlated with the temporal sequence of histologic changes, suggesting transcriptional regulation of gene expression during repair. Differences in gene expression between hard and soft callus and in each of these regions as repair progressed suggest local regulation of gene expression during cell differentiation and matrix synthesis.

Transforming growth factor beta 1 stimulates type II collagen expression in cultured periosteum-derived cells.

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.

Osteoblasts express types I and II activin receptors during early intramembranous and endochondral bone formation.

Increasing evidence suggests a potential role for activin in bone formation. However, the cognate receptors through which activins function with respect to skeletal tissues have not yet been identified. Identification and regulation of expression of these receptors are necessary prerequisites to understanding the role of activins in bone metabolism. We detected mRNAs for three activin receptors, type I (ActRI), type II (ActRII), and type IIB (ActRIIB), in multiple skeletal tissues in rat, including tibia and costochondral growth plate, and also in cultured osteoblasts. To gain information about the relationship between receptor expression and different skeletal cell functions, we evaluated expression of the three receptors in a semiquantitative manner during the early stages of fracture healing, a model for rapid bone formation. Relatively high levels of ActRI and ActRII expression were detected in the callus at 7, 10, and 14 days after fracture, times that correlate with the interval of rapid intramembranous bone formation and the initiation of endochondral bone formation. Expression of the ActRIIB in the fracture callus was strikingly lower than either ActRI or ActRII. Immunostaining of the fracture callus and the newborn rat femur with an anti-ActRII antibody localized the receptor to osteoblasts at regions of membranous and endochondral bone formation. No staining of osteoblasts in fracture callus or bone was seen with an anti-ActRIIB antibody. These results provide strong evidence of the identification of the principal receptors through which activins could function in the skeletal system and further shed light on activin's mechanism of action in bone formation.

Ultrasound-mediated transfection of mammalian cells.

Mammalian cells were successfully transfected with plasmid DNA in vitro using ultrasound transmitted through the walls of cell culture flasks or plates. Primary rat fibroblasts or chondrocytes were exposed to ultrasound in the presence of plasmids containing lacZ or neo genes. The transfection efficiency was evaluated by counting the number of beta-galactosidase (beta-Gal) positive cells or neomycin-resistant colonies. Transfection efficiency was optimized by varying ultrasound conditions, ambient temperatures (room temperature or 37 degrees C), plasmid concentrations, and initial cell populations. Additional experiments were performed performed to elucidate the mechanism of the ultrasound-mediated transfection. Maximal gene transfection was seen with two ultrasound conditions: 1-MHz carrier frequency 411 +/- 189 kPascal continuous wave with 20 or 30 sec of exposure time, and 1 MHz carrier frequency 319 +/- 157 kPascal continuous wave with 40 or 60 sec of exposure time. Gene expression was negligible when transfection procedures were performed at room temperature. The average stable transfection rate was 0.34% of surviving cells with a plasmid concentration of 40 micrograms/ml in primary fibroblasts. The transient transfection rate was 2.4% of surviving cells for primary chondrocytes. Data suggest that increasing plasmid concentration will increase efficiency. Identical treatment with 3.5 MHz produced no transfection, implying that cavitation produced by the ultrasound pressure wave appeared to play a critical role in mediating transfection. Ultrasound-mediated transfection was effective for suspended cells as well as for plated cells. This transfection method is simple, easy to keep sterile, and convenient. Ultrasound-mediated transfection appears to be a promising method for gene transfer into mammalian cells.

Induction of growth plate cartilage ossification by basic fibroblast growth factor.

In mammals, longitudinal bone growth results from the precise coupling of chondrogenesis and osteogenesis within the epiphyseal growth plate, a process termed endochondral ossification. The mechanisms coupling chondrogenesis and osteogenesis are unknown. Previous studies have shown that both basic fibroblast growth factor (bFGF) and acidic FGF are expressed by growth plate chondrocytes. Here we show that bFGF, infused directly into the rabbit proximal tibial growth plate, accelerates vascular invasion and ossification of growth plate cartilage. Our results suggest the possibility that bFGF (or a related member of the FGF family) couples osteogenesis to chondrogenesis by attracting vascular and bone cell invasion from the adjacent metaphyseal bone.

Differential mRNA fingerprinting by preferential amplification of coding sequences.

The need for rapid identification of differentially expressed genes will persist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquires expressed sequence tags (ESTs) from the extreme 3' non-coding end of mRNAs. Such ESTs have limitations for downstream applications. We have developed a method, termed preferential amplification of coding sequences (PACS), that was applied to identify differentially expressed coding sequence tags (dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequences in mRNAs and another set of primers to anchor at a PCR-amplifiable distance from AUG sequences. An initial screen identified 103 candidate dCSTs after screening approximately 15% of the expressed genes between the two cell types. Of these sequences, 27 represent CSTs of known genes and two are from 3'-ESTs of known mRNAs. Thus, PACS identified CSTs approximately 13.5 times more often than it identified 3' ESTs, attesting to the objective of the method. Since many of the dCSTs represent known genes, their identity and potential relevance to osteosarcoma could be immediately hypothesized. Differential expression of many of the dCSTs was further demonstrated by northern blotting or RT-PCR. Since PACS is not dependent on the existence of a poly A tail on an mRNA, it should have application to identify dCSTs for both prokaryotic and eukaryotic organisms. Additionally, PACS should aid in the identification of cell-specific or tissue-specific genes and bidirectional acquisition of cDNA sequence enabling rapid retrieval of full-length cDNA sequence of novel genes.

Identification of twenty-two candidate markers for human osteogenic sarcoma.

Since osteogenic sarcoma (OGS) predominantly affects children, its etiology and progression may be determined more by genetic than environmental factors. A few genes have been associated with OGS, however, their value in the diagnosis and/or prognosis of the disease remains poor. Evidently, more markers need to be identified for improving management of patients with OGS. To identify potential genetic markers for OGS, we have extended preferential amplification of coding sequences (PACS) to screen multiple samples simultaneously. The extended method is termed multi-PACS. Multi-PACS was applied between a normal osteoblast and four OGS-derived cell lines to identify differentially expressed coding sequence tags (dCST) that identified 145 dCSTs. Subsequently, differential mRNA expression was validated for a chosen subset of 22 dCSTs. These chosen dCSTs include among others cyclins D and E, two cyclin dependent kinases, two other kinases, transcription factors E2F4, E2F5, and p130, a DNA repair gene, a gene for the signalosome subunit, and potential guanine nucleotide binding factors. We infer that these genes could be so easily identified because PACS preferentially identifies coding instead of non-coding sequences. We also infer that these genes identify signaling pathways pertinent to OGS. mRNA expression profile of these 22 genes/dCSTs generated distinct expression signature of the OGS-derived cell lines suggesting that further work on clinical samples with these dCSTs will yield valuable information for OGS. We conclude that these 22 genes/dCSTs are candidate markers for OGS.

Improved design of riboprobes from pBluescript and related vectors for in situ hybridization.

The pBluescript family of plasmids and phagemids are sophisticated multi-purpose cloning vectors that allow convenient production of single-stranded sense and anti-sense RNA probes corresponding to DNA sequences inserted into a large multiple cloning site array. We have observed that in many applications sense (control) probes generated from genes cloned into pBluescript II KS(-) give high background signals on in situ hybridization to human tissue sections. Our studies indicate that this spurious hybridization is due to sequences contained within both strands of the multiple cloning site between the SmaI and SacI sites that are similar to human 28S rRNA. This information is useful in construct design in order to minimize nonspecific background problems, as demonstrated by in situ hybridization of sense and anti-sense probes corresponding to a portion of human stromelysin-3 to sections of human lung carcinoma.

Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR.

The determination of unknown DNA sequences around a known locus has important applications in molecular genetics, specifically in genomic walking and genome mapping. Several PCR-based methods have been reported to address this issue, but they often involve multiple, time-consuming steps. We have previously described a technique known as restriction site PCR (RS-PCR) that allows sequence acquisition faster than the existing methods. The method involves PCR using four separate universal primers that are representative of given restriction enzyme sites (RS primers), and a specific primer from one end of the known sequence. We have now significantly improved the technique by mixing the four universal primers into one PCR tube with the first specific primer. This is followed by a nested PCR with the mixed RS primers and an internal specific primer, after which the product is sequenced by direct automated sequencing. The technique, called multiplex RS-PCR (mRS-PCR), is reproducible and can be used to obtain unknown sequence adjacent to known sequences in both the upstream and downstream directions. We illustrate the application of mRS-PCR in the acquisition of approximately 780 bp of genomic sequence starting from a known sequence of approximately 120 bp. Multiplex RS-PCR appears to be the fastest of all methods that address the issue of unknown sequence retrieval adjacent to a known region.

Gene identification by arrested primer extension.

This paper reports a novel method for the identification of nucleic acid target sequences when these targets have high sequence identity. Homologous genes are currently identified by sequencing. We hypothesize that by primer extension in the presence of selected nucleotides, genes with similar sequence can be identified by the length of the extension products on gel electrophoresis. This simple procedure eliminates the much-demanding process of sequencing. We term this process Arrested Primer Extension (APE). As a demonstration of the feasibility of this method, we have used APE to speciate a known set of cultured mycobacteria. There should be many other applications of this method.

Detection and measurement of simulated early rheumatoid lesions of the hand using digital subtraction radiography.

The ability of digital subtraction radiography, a new technique to detect and quantify small bone lesions, is demonstrated. Discrete lesions in the metacarpals of cadaver hands simulated erosive bone loss. Radiographs made before and after removal of bone were digitized and subtracted. Density changes on subtraction images were determined, and bone loss was estimated by an automatic procedure that compared changes in radiographic density with a calibration wedge included in the radiographs. Comparison of estimated bone loss with the weight of bone removed showed reproducible detection and measurement of bone lesions as small as 4.6 mg, a size undetectable using current radiographic methods. Subtraction radiographs of bone chips overlaid on the hand of a volunteer indicated detection limits were similar in vivo. This technique enhanced the radiographic visibility of erosive lesions and thus has the potential to improve the detection of subtle bone changes in clinical settings.

Direct sequencing of unpurified PCR-amplified DNA by semi-exponential cycle sequencing (SECS).

A simple technique for direct sequencing of PCR-amplified templates without purification of the PCR reaction product is presented. This method does not require an additional synthesis step after template amplification, and can generate sequence information form as little as 0.1 fmol of unpurified template.

Selective differential fingerprinting. A method for identifying differentially expressed genes in a family between two samples.

A method termed selective differential fingerprinting (SDF) has been developed that enables one to investigate the level of expression for a family of genes between two samples. SDF produces a fingerprint (on a sequencing gel) on reverse transcription polymerase chain reaction (RT-PCR) of a sample with degenerate primers designed from conserved regions of a family of genes. By comparing fingerprints obtained after SDF with primers representing the transforming growth factor-beta (TGF beta) family of growth factors between a low-grade and a high-grade tumor from the same patient, a TGF beta family member known as osteogenic protein 1 (OP-1) or bone morphogenic protein 7 (BMP-7) was found to be greatly overexpressed in the high-grade tumor compared to the low-grade one. SDF also has the potential to identify novel genes. SDF offers a general way to identify differentially expressed genes for a family between two given samples.

PIG-B: a homemade monophasic cocktail for the extraction of RNA.

An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent are Phenol, Isoamyl alcohol, Guanidinium isothiocyanate, and Beta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.

An effective method of completely removing contaminating genomic DNA from an RNA sample to be used for PCR.

A simple method for removing contaminating genomic DNA from an RNA preparation is presented. The method involves digestion of the RNA with RNase-free DNase I at room temperature followed by inactivation of the enzyme at 65 degrees C in presence of EDTA. This method produces an RNA sample that is negative for genomic DNA by PCR.

Gene digging. A method for obtaining species-specific sequence based on conserved segments of nucleotides in open reading frames.

A method termed "gene digging" has been developed based on our observation of stretches of highly conserved nucleotide sequence in the coding region of many genes across related species. Rabbit-specific nucleotide sequences corresponding to desired coding segments of 14 different genes were obtained with primers that were designed based on conserved nucleotide stretches. Our success in gene digging could be attributable to the method's inherent ability to reduce the degeneracy of primers by more than two orders of magnitude (sometimes by more than three orders of magnitude) compared to primers designed from conserved amino acids. Our results not only demonstrate the value of the method, but also hint at a thus far unknown functional significance of conserved nucleotide stretches in the coding region of various genes. In our hands the method worked 14 out of 14 times indicating generality of the concept.