Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background

In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.

Methods

Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.

Results

Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.

Conclusion

Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.     

Polymorphism in 5′ untranslated region of heat‐shock protein 70 gene as marker of post‐partum anoestrus in Murrah buffaloes

The enormous production potential of buffaloes has never been accomplished due to various reproductive insufficiencies. Among them, post‐partum anoestrus, a multifactorial disorder, is predominant but any genetic association is yet to be established. This study focused to identify novel polymorphisms in heat‐shock protein 70 (HSP70) gene and its possible association with post‐partum anoestrus in Murrah buffaloes. A 579‐bp fragment from 5′ untranslated region of HSP70 gene was amplified by polymerase chain reaction from blood genomic DNA of 614 animals maintained under similar management conditions. In phase‐I experiment, custom sequencing and restriction enzyme (RE) digestion of the amplified fragment were performed in 40 buffaloes with similar post‐partum oestrous conditions over previous consecutive three or more gestations—20 animals each showing post‐partum anoestrus (>120 days after parturition) and normal cyclicity (<65 days after parturition). While in phase‐II experiment, herd screening by RE analysis was performed in remaining 574 animals. Four transversions at T‐75G, C+31G, T+38G and C+97A and three transition mutations at T‐153C, T+33C and A+44G positions were observed. Polymorphism at T+38G site revealed significant (p < .05) variation, where homozygous G was present only in post‐partum anoestrous animals while nucleotide T was present randomly in both groups of phase‐I animals whereas phase‐II experiments revealed homozygous G in 55 animals. Regression analysis in relation to average post‐partum interval against genotypic frequencies at T+38G also depicted significant association. HSP70 gene polymorphism at T+38G position can therefore be used as genetic marker for excluding probable post‐partum anoestrous buffaloes from herd for breeding programmes.