Differential gene expression in peripheral blood lymphocytes of cancer patients treated with whole body hyperthermia and chemotherapy: A pilot study

Figures & data

Table I.  Genes induced by whole body hyperthermia and chemotherapy in peripheral blood lymphocytes of patients with metastatic adenocarcinomas and the reference gene GAPDH.

Gene	Accession^#	GI	Primer sequence (5′ → 3′)
HSP27^a	AB020027	11036356	Sense	5′-gTC CCT ggA TgT CAA CCA CT-3′
			Antisense	5′-ATC ggA TTT TgC AgC TTC Tg-3′
HSP60	BC002676	GI:12803680	Sense	5′-Tgg TgC AgT gTT Tgg AgA Ag-3′
			Antisense	5′-ATg AgT CCA Agg CTg gAA Tg-3′
HSP70^a	M11717	GI:184416	Sense	5′-ACA ggC Tgg TgA ACC ACT TC-3′
			Antisense	5′-Agg ACC Agg TCg TgA ATC Tg-3′
HSP90a	NM005348	GI:13129149	Sense	5′-Tgg TgA CAT CTC CAT gCT gT-3′
			Antisense	5′-CgT gAT gTg TCg TCA TCT CC-3′
HSP90b	XM018115	GI:13654496	Sense	5′-TAg AAg AgA ggC ggg TCA AA-3′
			Antisense	5′-TCT ggT CCA AAT Agg CTT gg-3′
HSP105	AB003334	GI:3970830	Sense	5′-gCC CAA AAT AAA ggT ggT gA-3′
			Antisense	5′-Tgg TCC ACA CAg CTT gTC TC-3′
CaCyBP	AF314752	GI:11321440	Sense	5′-TgT ggT TgC TCC CAT TAC AA-3′
			Antisense	5′-CCC ACC TTg TgT TTT CCA CT-3′
MIP-1beta	NM002984	GI:4506844	Sense	5′-gAg TTC TgC AgC CTC ACC TC-3′
			Antisense	5′-ACC ACA AAg TTg CgA ggA Ag-3′
HBB	NM000518	GI:13788565	Sense	5′-TTg gAC CCA gAg gTT CTT Tg-3′
			Antisense	5′-gCC ACC ACT TTC TgA TAg gC-3′
GAPDH	M33197	GI:182976	Sense	5′-ACC ACA gTC CAT gCC ATC AC-3′
			Antisense	5′-TCC ACC ACC CTg TTg CTg TA-3′

cDNAs induced by whole body hyperthermia (WBH) were identified using suppression subtractive hybridization (SSH) and by semi-quantitative RT-PCR (sq-RT-PCR) from peripheral blood lymphocytes (PBL) of patients with metastatic tumours isolated before and after WBH. Shown are the names of genes, accession numbers, GI and primer sequences used for sq-RT-PCR. All primers were generated using the program Primer3 (www.genome.wi.mit.edu). except for GAPDH (Clontech).

^aDifferential expression pattern of HSP27 and HSP70 was detected by sq-RT-PCR only.

Figure 1. Dot-blot hybridization of cDNA library enriched for cDNAs upregulated in peripheral blood lymphocytes after whole body hyperthermia probed with PBL cDNAs increased after (after WBH) and before whole body hyperthermia combined with chemotherapy (before WBH), respectively. Marked are clones of cDNAs that are increased ((a)1–3), as well as cDNAs with low (b) and high initial signal intensity (c) that are not differentially expressed.

Figure 2. Semi-quantitative RT-PCR (sqRT-PCR) with 1:10 diluted second suppressive PCR material as template generated from combined RNAs from all patients, −: before and +: after WBH with simultaneous chemotherapy, M: 100 bp ladder. Shown are identical cycles for every gene tested. Genes induced are shown (HSP60, HSP90a, HSP90b, HSP105, CacyBP, MIP-1 beta, HBB and the house keeping gene GAPDH for control).

Figure 3. Densitometric evaluation of sqRT-PCR with cDNA of four different patients undergoing WBH and chemotherapy (lymphocyte samples before and after treatment). Shown is the relative increase of expression after therapy (×fold ± SD) of HSP27, HSP60, HSP70, HSP90a, HSP90b, HSP105, CacyBP, MIP-1 beta, HBB.